Oct3/4, Klf4, c-Myc and Sox2 produce induced pluripotent stem cells

ABSTRACT

The present invention relates to a nuclear reprogramming factor having an action of reprogramming a differentiated somatic cell to derive an induced pluripotent stem (iPS) cell. The present invention also relates to the aforementioned iPS cells, methods of generating and maintaining iPS cells, and methods of using iPS cells, including screening and testing methods as well as methods of stem cell therapy. The present invention also relates to somatic cells derived by inducing differentiation of the aforementioned iPS cells.

PRIOR RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/213,035, filed Jun. 13, 2008, which is a continuation-in-part ofPCT/JP2006/324881, filed Dec. 6, 2006, which claims priority to JapaneseApplication No. 2005-359537, filed Dec. 13, 2005, and this applicationis a continuation of U.S. patent application Ser. No. 12/213,035, filedJun. 13, 2008, which claims priority to U.S. Provisional Application No.61/001,108, filed Oct. 31, 2007, and U.S. Provisional Application No.60/996,289, filed Nov. 9, 2007. The entire disclosures of each of theabove-cited applications are considered as being part of thisapplication and are expressly incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

The present invention relates to a nuclear reprogramming factor havingan action of reprogramming a somatic cell to derive an inducedpluripotent stem (iPS) cell. The present invention also relates to theaforementioned iPS cells, methods of generating and maintaining iPScells, and methods of using iPS cells, including screening and testingmethods as well as methods of stem cell therapy. The present inventionalso relates to somatic cells derived by inducing differentiation of theaforementioned iPS cells.

BACKGROUND OF THE INVENTION

Embryonic stem cells (ES cells) are stem cells established from human ormouse early embryos which have a characteristic feature that they can becultured over a long period of time while maintaining pluripotentability to differentiate into all kinds of cells existing in livingbodies. Human embryonic stem cells are expected for use as resources forcell transplantation therapies for various diseases such as Parkinson'sdisease, juvenile diabetes, and leukemia, taking advantage of theaforementioned properties. However, transplantation of ES cells has aproblem of causing rejection in the same manner as organtransplantation. Moreover, from an ethical viewpoint, there are manydissenting opinions against the use of ES cells which are established bydestroying human embryos.

Embryonic stem (ES) cells, derived from the inner cell mass of mammalianblastocysts, have the ability to grow indefinitely while maintainingpluripotency (Evans et al., Nature 292:154-156, 1981; Martin, P.N.A.S.USA 78:7634-7638, 1981). These properties have led to expectations thathuman ES cells might be useful to understand disease mechanisms, toscreen effective and safe drugs, and to treat patients of variousdiseases and injuries, such as juvenile diabetes and spinal cord injury(Thomson et al., Science 282:1145-1147, 1998). Use of human embryos,however, faces ethical controversies that hinder the applications ofhuman ES cells. In addition, it is difficult to generate patient- ordisease-specific ES cells, which are required for their effectiveapplication. Therefore, if dedifferentiation of a patient's own somaticcells could be induced to establish cells having pluripotency and growthability similar to those of ES cells (in this specification, these cellsare referred to as “induced pluripotent stem cells (iPS cells)”, thoughthey are sometimes called “embryonic stem cell-like cells” or “ES-likecells”), it is anticipated that such cells could be used as idealpluripotent cells, free from rejection or ethical difficulties.

Methods for nuclear reprogramming of a somatic cell nucleus have beenreported. One technique for nuclear reprogramming which has beenreported involves nuclear transfer into oocytes (Wakayama et al., Nature394:369-374, 1998; Wilmut et al., Nature 385:810-813, 1997). Anothermethod, for example, a technique of establishing an embryonic stem cellfrom a cloned embryo, prepared by transplanting a nucleus of a somaticcell into an egg, was reported (Hwang et al., Science 303:1669-74, 2004;Hwang et al., Science 308:1777-83, 2005): these articles were, however,proved to be fabrications and later withdrawn. Others have reportedtechniques for nuclear reprogramming of a somatic cell nucleus by fusinga somatic cell and an ES cell (Tada et al., Curr. Biol. 11:1553-1558,2001; Cowan et al., Science 309:1369-73, 2005). Another reportedtechnique for reprogramming a cell nucleus involves treatment of adifferentiated cell with an undifferentiated human carcinoma cellextract (Taranger et al., Mol. Biol. Cell 16:5719-35, 2005). However,these methods all have serious drawbacks. Methods of nuclear transferinto oocytes and techniques which involve the fusion of ES anddifferentiated cells both comprise the use of ES cells, which presentethical problems. In addition, cells generated by such methods oftenlead to problems with rejection upon transplantation into an unmatchedhost. Furthermore, the use of cell extracts to treat differentiatedcells is technically unreliable and unsafe, in part because the cellextract components responsible for the nuclear programming are mixed insolution with other unknown factors.

A method for screening a nuclear reprogramming factor having an actionof reprogramming differentiated somatic cells to derive inducedpluripotent stems cell was proposed in International PublicationWO2005/80598, which is incorporated by reference in its entirety. Thismethod comprises the steps of: contacting somatic cells containing amarker gene under expression regulatory control of an ECAT (ES cellassociated transcript) gene expression control region with a testsubstance; examining presence or absence of the appearance of a cellthat expresses the marker gene; and choosing a test substance inducingthe appearance of said cell as a candidate nuclear reprogramming factorfor somatic cells. A method for reprogramming a somatic cell isdisclosed in Example 6 and the like of the above publication. However,this publication fails to report an actual identification of a nuclearreprogramming factor.

In view of these problems, there remains a need in the art for nuclearreprogramming factors capable of generating pluripotent stem cells fromsomatic cells. There also remains a need for pluripotent stem cells,which can be derived from a patient's own somatic cells, so as to renderethical issues and avoid problems with rejection. Such cells would haveenormous potential for both research and clinical applications.

SUMMARY OF THE INVENTION

The present invention provides induced pluripotent stem (iPS) cellsderived by nuclear reprogramming of a somatic cell. The presentinvention also provides methods for reprogramming of a differentiatedcell without using eggs, embryos, or embryonic stem (ES) cells. Thepresent invention also provides nuclear reprogramming factors forinduction of pluripotent stem cells. The disclosed methods and nuclearreprogramming factors may be used to conveniently and highlyreproducibly establish iPS cells having pluripotency and growth abilitysimilar to that of ES cells. More specifically, the present inventionprovides for inducing reprogramming of a differentiated cell withoutusing eggs, embryos, or ES cells to conveniently and highly reproduciblyestablish the iPS cells having pluripotency and growth ability similarto that of ES cells.

The invention provides a pluripotent stem cell induced by reprogramminga somatic cell in the absence of eggs, embryos, or embryonic stem (ES)cells. The somatic cell can be a mammalian cell, for example a mousecell or a human cell. The present invention also provides such apluripotent stem cell, wherein the reprogramming comprises contactingthe somatic cell with a nuclear reprogramming factor.

The nuclear reprogramming factor can comprise at least one gene product,for example a protein. The nuclear reprogramming factor can comprise agene product of an Oct family gene, a Klf family gene, a Myc familygene, or a Sox family gene. The nuclear reprogramming factor cancomprise one or more gene products of each of: an Oct family gene, a Klffamily gene, and a Sox family gene. The nuclear reprogramming factor cancomprise one or more gene products of each of: an Oct family gene, a Klffamily gene, a Myc family gene, and a Sox family gene. Furthermore, thenuclear reprogramming factor can comprise one or more gene products ofeach of: an Oct family gene, a Klf family gene, together with acytokine. The cytokine can be at least one of basic fibroblast growthfactor (bFGF) and stem cell factor (SCF).

The invention also provides a method for preparing an inducedpluripotent stem cell by nuclear reprogramming of a somatic cell, whichcomprises contacting a nuclear reprogramming factor with the somaticcell to obtain an induced pluripotent stem cell. The invention alsoprovides such a method which is performed in the absence of eggs,embryos, or embryonic stem (ES) cells. The present invention alsoprovides an induced pluripotent stem cell obtained by such a method. Thepresent invention also provides a pluripotent stem cell induced byreprogramming a somatic cell, wherein the reprogramming comprisescontacting the somatic cell with a nuclear reprogramming factor.

The present invention also provides such a method wherein the nuclearreprogramming factor comprises one or more gene products of each of: anOct family gene, a Klf family gene, and a Sox family gene. The presentinvention also provides such a method wherein the nuclear reprogrammingfactor comprises one or more gene products of each of: Oct3/4, Klf4, andSox2. The present invention also provides such a method wherein thenuclear reprogramming factor further comprises one or more gene productsof a Sall4 gene. The present invention also provides pluripotent stemcells prepared by such methods.

The present invention also provides such a method wherein the nuclearreprogramming factor comprises one or more gene products of each of:wherein the nuclear reprogramming factor comprises one or more geneproducts of each of: an Oct family gene, a Klf family gene, a Myc familygene, and a Sox family gene. The present invention also provides such amethod wherein the nuclear reprogramming factor comprises one or moregene products of each of: Oct3/4, Klf4, c-Myc, and Sox2. The presentinvention also provides such a method wherein the nuclear reprogrammingfactor further comprises one or more gene products of a Sall4 gene. Thepresent invention also provides pluripotent stem cells prepared by suchmethods.

The present invention also provides such a method wherein the nuclearreprogramming factor comprises one or more gene products of each of:Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28. The present invention alsoprovides pluripotent stem cells prepared by such a method.

The present invention also provides a method of inducing a somatic cellto become a pluripotent stem cell comprising contacting the somatic cellwith a nuclear reprogramming factor under conditions to obtain apluripotent stem cell free of rejection.

The present invention also provides a somatic cell derived by inducingdifferentiation of an induced pluripotent stem cell as disclosed herein.

The present invention also provides a method for stem cell therapycomprising: (1) isolating and collecting a somatic cell from a patient;(2) inducing said somatic cell from the patient into an iPS cell (3)inducing differentiation of said iPS cell, and (4) transplanting thedifferentiated cell from (3) into the patient.

The present invention also provides a method for evaluating aphysiological function of a compound comprising treating cells obtainedby inducing differentiation of an induced pluripotent stem cell asdisclosed herein with the compound.

The present invention also provides a method for evaluating the toxicityof a compound comprising treating cells obtained by inducingdifferentiation of an induced pluripotent stem cell as disclosed hereinwith the compound.

Other features and advantages of the present invention will be set forthin the description of the invention that follows, and will be apparent,in part, from the description or may be learned by practice of theinvention. The invention will be realized and attained by thecompositions, products, and methods particularly pointed out in thewritten description and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a screening method for reprogramming factors usingembryonic fibroblasts (MEFs) of a mouse having βgeo knock-in Fbx15 gene.

FIG. 2 depicts photographs showing morphology of iPS cells obtained byintroducing the 24 genes shown in TABLE 4. Morphologies ofdifferentiated cells (MEF) and of normal embryonic stem cells (ES) arealso shown as a reference.

FIG. 3 shows expression profiles of marker genes in iPS cells. Theresults of RT-PCR using total RNAs extracted from iPS cells, ES cellsand MEF cells as templates are shown.

FIG. 4 shows methylation status of DNA in iPS cells. Genomic DNAsextracted from iPS cells, ES cells, and MEF cells were treated withbisulfite. The target DNAs were amplified by PCR and then inserted intoplasmid. Ten clones of plasmid were isolated for each of the genes, andsequenced. Methylated CpGs are indicated with closed circles, andunmethylated CpGs with open circles.

FIG. 5 shows colony numbers of G418-resistant cells obtained bytransduction of 24-gene group and 23-gene groups wherein each individualgene was withdrawn from the 24-gene group. The lower parts of the graphshow colony numbers obtained in one week after the G418 selection, andthe upper parts of the graph show numbers of clones obtained in threeweeks. When each boxed gene (the reference number for each gene is thesame as that indicated in TABLE 4) was withdrawn, no colonies wereobtained at all, or only a few colonies were observed after 3 weeks.

FIG. 6 shows colony numbers of G418-resistant cells obtained bytransduction of 10-gene group and 9-gene groups wherein each individualgene was withdrawn from the 10-gene group. When each of genes #14, #15or #20 was withdrawn, no colony was obtained. When gene #22 waswithdrawn, a few G418-resistant colonies were obtained. However, thecells gave differentiated morphology which was apparently different fromthat of iPS cells.

FIG. 7 shows numbers of G418-resistant emerged colonies (reprogrammedcolony) with 10-gene group, 4-gene group, 3-gene groups, or 2-genegroups. Typical morphology and sizes of the colonies are shown.

FIG. 8 depicts photographs showing results of hematoxylin-eosin (H & E)staining of tumors formed after subcutaneous transplantation of iPScells derived from MEFs into nude mice. Differentiation into a varietyof tissues in a triploblastic system was observed.

FIG. 9 depicts photographs of embryos prepared by transplanting iPScells derived from adult dermal fibroblasts into mouse blastocysts andtransplanting the cells into the uteri of pseudopregnant mice. It can beobserved that, in the upper left embryo, cells derived from the iPScells (emitting green fluorescence) were systemically distributed. Inthe lower photographs, it can be observed that almost all cells of theheart, liver, and spinal cord of the embryo were GFP-positive and werederived from the iPS cells.

FIG. 10 depicts photographs showing results of RT-PCR confirming theexpression of the ES cell marker genes. In the photographs, Sox2 minusindicates iPS cells established by the transduction of 3 genes intoMEFs, 4ECATs indicates iPS cells established by the transduction of 4genes into MEFs, 10ECATs indicates iPS cells established by thetransduction of 10 genes into MEFs, 10ECATs Skin fibroblast indicatesiPS cells established by the transduction of 10 genes into dermalfibroblasts, ES indicates mouse ES cells, and MEF indicates MEF cellswithout gene transduction. The numerical values under the symbolsindicate clones numbers.

FIG. 11 shows an effect of bFGF on the establishment of iPS cells fromMEFs. Four factors (upper row) or three factors except for c-Myc (lowerrow) were retrovirally transduced into MEFs derived fromFbx15^(62 geo/βgeo) mice, and cultured on ordinary feeder cells (STOcells) (left) and bFGF expression vector-introduced STO cells (right).G418 selection was performed for 2 weeks, and cells were stained withcrystal blue and photographed. The numerical values indicate the numberof colonies.

FIGS. 12(A)-(B) depict explanations of the experiments usingNanog-EGFP-IRES-Puro^(r) mice. (A) E. coli artificial chromosome (BAC)containing the mouse Nanog gene in the center was isolated, and theEGFP-IRES-Puro^(r) cassette was inserted upstream from the coding regionof Nanog by recombineering. (B) Transgenic mice were prepared with themodified BAC. GFP expression was observed limitedly in inner cell massesof blastocysts and gonads.

FIG. 13 depicts explanations of the experiments usingNanog-EGFP-IRES-Puro^(r) mice. From embryos of Nanog-EGFP-IRES-Puro^(r)mice (13.5 days after fertilization), heads, viscera and gonads wereremoved to establish MEFs. As a result of analysis with a cell sorter,almost no GFP-positive cells existed in MEFs derived from theNanog-EGFP-IRES-Puro mice (Nanog) in the same manner as theFbx15^(βgeo/βgeo) mouse-derived MEFs (Fbx15) or wild-type mouse-derivedMEFs (Wild).

FIG. 14 depicts photographs of iPS cells established from theNanog-EGFP-IRES-Puro mouse MEFs (left) and the Fbx15^(βgeo/βgeo) mouseMEFs (right). The cells were selected with puromycin and G418,respectively.

FIG. 15 shows results of growth of iPS cells. 100,000 cells of each ofES cells, iPS cells derived from the Nanog-EGFP-IRES-Puro mouse MEFs(Nanog iPS, left), and iPS cells derived from the Fbx15^(βgeo/βgeo)mouse MEFs (Fbx iPS) were seeded on 24-well plates, and passaged every 3days. Cell count results are shown. The numerical values representaverage doubling times.

FIG. 16 shows gene expression profiles of iPS cells. Expression of themarker genes in MEFs, ES cells, iPS cells derived fromNanog-EGFP-IRES-Puro mouse MEFs (Nanog iPS, left), and iPS cells derivedfrom Fbx15^(βgeo/βgeo) mouse MEFs (Fbx iPS) were analyzed by RT-PCR. Thenumerical values at the bottom indicate the numbers of passages.

FIG. 17 shows teratoma formation from the Nanog iPS cells. 1,000,000cells of each of ES cells or Nanog iPS cells #24 (passage 8 times) weresubcutaneously injected into the backs of nude mice, and the appearanceof tumors formed after 3 weeks (left) and tissue images (right, H & Estained) are shown.

FIG. 18 shows preparation of chimeric mice with the Nanog iPS cells. Thechimeric mice that were born after transplantation of the Nanog iPScells (clone NPMF4EK-24, passaged 6 times) into the blastocysts. Fourchimeric mice were born from 90 transplanted embryos.

FIG. 19 shows germ-line transmission from the Nanog iPS cells. PCRanalysis of genomic DNA of mice, born by mating of the chimeric miceshown in FIG. 18 and C57BL/6 mice, revealed the existence of transgenesof Oct3/4 and Klf4 in all of the mice, thereby confirming germ-linetransmission.

FIG. 20 shows induction of differentiation into nerve cells from iPScells. Nerve cells (top, βIII tubulin-positive), oligodendrocytes (left,O4-positive), and astrocytes (right, GFAP-positive) differentiated invitro from dermal fibroblasts-derived iPS cells are shown.

FIG. 21 depicts explanations of establishment of the iPS cells withoutusing drug selection. MEFs at 10,000 to 100,000 cells per 10 cm dishwere seeded, and the 4 factors were retrovirally transduced. No colonyappeared in the control (Mock, top left), whilst in the dish with thetransduction by the 4 factors, swelling colonies similar to those of theiPS cells were obtained (bottom left and center), as well as flattransformant colonies. When the cells were passaged, cells similar tothe iPS cells were obtained (right).

FIG. 22 shows gene expression profiles of cells established withoutusing drug selection. RNA was extracted from the established cells shownin FIG. 21, and expression of the ES cell marker genes was analyzed byRT-PCR.

FIG. 23(A)-(B) show iPS cell-like cells derived from human fibroblasts.The colonies obtained by retroviral transduction with human homologousgenes of the 4 factors into fibroblasts derived from human embryos (FIG.23(A)), and the cells after two passages (FIG. 23(B)) are shown.

FIG. 24 shows establishment of the iPS cells from human adult dermalfibroblasts. The factors mentioned in the left column were transducedretrovirally into human adult dermal fibroblasts infected with the mouseretroviral receptor with lentivirus. The photographs shows phasecontrast images (object×10) on day 8 after the viral infection.

FIGS. 25(A)-(B) show the results of alkaline phosphatase staining of iPScells from two different experiments. (A) 8×10⁵ HDFs derived from adultskin expressing mouse Slc7a1 gene and introduced with pMXs encoding thegenes indicated were plated on mitomycin C-treated STO cells. Theinfected cells were cultured in ES medium for 12 days. The cells werestained with alkaline phosphatase. (B) BJ fibroblasts expressing mouseSlc7a1 gene were plated at 8×10⁵ cells per 100 mm dish on mitomycin Ctreated STO cells. Next day, the cells were transduced with the genesindicated (left) by retroviral infection. After transduction, the cellswere maintained in ES medium for 2 weeks. After picking up the colonies,the cells were stained with alkaline phosphatase.

FIG. 26(A)-(B) show Cyanine 3 (Cy-3) staining of iPS (-like) cells withES cell markers. (A) iPS (-like) cells derived from adult human dermalfibroblasts (HDFs) were plated at 5×10⁴ cells per well of 6 well plateson mitomycin C-treated STO cells, and grown for 4 days. The cells werefixed with PBS containing 10% formalin, and blocked with blocking buffer(0.1% bovine serum albumin and 10 mg/ml normal donkey serum in PBS) for45 minutes at room temperature. Primary antibodies indicated above werediluted 1:100 in blocking buffer. Overnight incubation with primaryantibody, the cells were washed with PBS, and then incubated withsecondary antibody. Cy-3-conjugated anti-mouse IgG (for ABCG-2 andSSEA-4) and anti-rat IgM (for SSEA-3) antibodies were used. (B) iPS(-like) cells derived from adult HDFs (clone 87E3, 87E4 and 87E12) wereplated at 5×10⁴ cells per well of a 6 well plate on mitomycin C-treatedSTO cells, and grown for 5 days. Parental HDFs also plated on 6 wellplate and maintained for 2 days. The cells were fixed with PBScontaining 10% formalin, and blocked with blocking buffer (3% BSA inPBS) for 45 minutes at room temperature. Primary antibodies indicatedabove were diluted 1:100 in blocking buffer. Overnight incubation withprimary antibody, the cells were washed with PBS, and then incubatedwith secondary antibody. Cy-3-conjugated anti-mouse IgG (for ABCG-2,E-cadherin, and SSEA-4) and anti-rat IgM (for SSEA-3) antibodies wereused as secondary antibodies.

FIG. 27 shows human iPS (-like) cells express ECATs. Total RNA wasisolated from human iPS (-like) cells (iPS-HDFaSlc-87E-1˜8, 11 and 12),NTERA2 cloneD1 human embryonic carcinoma cells (passage 35) and adultHDFs expressing mouse Slc7a1 gene (passage 6). First-strand cDNA wassynthesized by using oligo-dT20 primer and Rever Tra Ace-α-kit (Toyobo)according to manufacturer's protocol. PCR was performed with the primersas follows: hOct4 S1165 and hOct4-AS1283 for endogenous OCT4,hSox2-S1430 and hSox2-AS1555 for endogenous SOX2, ECAT4-macaca-968S andECAT4-macaca-1334AS for NANOG, hRex1-RT-U and hRex1-RT-L for REX1,hFGF4-RT-U and hFGF4-RT-L for FGF4, hGDF3-S243 and hGDF3-AS850 for GDF3,hECAT15-S532 and hECAT15-AS916 for ECAT15-1, hECAT15-2-S85 andhECAT15-2-AS667 for ECAT15-2, hpH34-S40 and hpH34-AS259 for ESG1,hTERT-S3556 and hTERT-AS3713 for hTERT, and G3PDH-F and G3PDH-R forG3PDH.

FIG. 28 shows human iPS (-like) cells express ECATs. Total RNA wasisolated from human iPS (-like) cells (iPS-BJSlc-97E-1, 2, 4, 5, 6, 7,8, 10, 11, 12, -97G-3, 5, -97H-3, 5), NTERA2 clone D1 human embryoniccarcinoma cells (passage 35) and BJ fibroblasts expressing mouse Slc7a1gene (passage 6). First-strand cDNA was synthesized by using oligo-dT20primer and Rever Tra Ace-α-kit (Toyobo) according to manufacturer'sprotocol. PCR was performed with the primers as follows: hOct4 S1165 andhOct4-AS1283 for endogenous OCT4, hSox2-S1430 and hSox2-AS1555 forendogenous SOX2, ECAT4-macaca-968S and ECAT4-macaca-1334AS for NANOG,hRex1-RT-U and hRex1-RT-L for REX1, hFGF4-RT-U and hFGF4-RT-L for FGF4,hGDF3-S243 and hGDF3-AS850 for GDF3, hECAT15-S532 and hECAT15-AS916 forECAT15-1, hECAT15-2-S85 and hECAT15-2-AS667 for ECAT15-2, hpH34-S40 andhpH34-AS259 for ESG1, hTERT-S3556 and hTERT-AS3713 for hTERT, andG3PDH-F and G3PDH-R for G3PDH.

FIGS. 29(A)-(D) show teratoma formation. Five million of hiPS (-like)cells were subcutaneously injected into dorsal flanks of SCID mouse(female, 5 weeks old). Two months after injection, large tumors wereobserved. Tumors were dissected, weighed and photographed. Then thesetumors were fixed with PBS containing 10% formalin. Paraffin-embeddedtumor was sliced and then stained with hematoxylin and eosin. (A) Mousefrom clone iPS-HDFa/Slc-87E-12. (B)-(D) indicate mouse teratomas fromclones iPS-HDFa/Slc-97E-3 (B); iPS-HDFa/Slc-87E-6(C); andiPS-HDFa/Slc-87E-12 (D).

FIG. 30 shows in vitro differentiation of human iPS-like cells. Thecells (iPS-HDFaSlc-127F2, E3) were suspended in hES medium (w/o bFGF).2×10⁶ cells were transferred to HEMA (2-hydroxyethylmethacrylate)-coated 100 mm tissue culture dish. The medium was changedevery other day. After seven days floating culture, the cells werecollected, plated to six gelatinized 35 mm dishes and incubated another7 days. The cells were fixed with PBS containing 10% formalin for 10 minat room temperature, permeabilized with PBS containing 0.5% TritonX-100for 5 min at room temperature, and blocked with PBS containing 3% BSAfor 30 min at room temperature. Primary antibodies used in thisexperiment were as follows; anti-α-smooth muscle actin (Ms mono,pre-diluted, DAKO), anti-βIII-tubulin (Ms mono, 1:100 in blockingbuffer, Chemicon), anti-α-fetoprotein (Rb poly, pre-diluted, DAKO),normal mouse IgG (2 mg/ml, Chemicon), and normal rabbit IgG (2 mg/ml,Chemicon). After incubation with primary antibody (1 hour at roomtemperature), the cells were washed with PBS, and incubated withsecondary antibody (1:300 in blocking buffer).

FIG. 31 shows improved transduction efficiency of retroviruses in humanHDFs. HDFs or HDFs expressing mouse Slc7a1 gene (HDF-Slc7a1) wereintroduced with ecotropic (Eco) or amphotropic (Ampho) pMX retrovirusescontaining the GFP cDNA. Shown are results of fluorescent microscope(upper) and flow cytometry (lower). Bars=100 μm.

FIGS. 32(A)-(N) show induction of iPS cells from adult HDFs in primateES cell media. (A) Time schedule of iPS cell generation. (B) Morphologyof HDFs. (C) Typical image of non-ES cell-like colony. (D) Typical imageof hES cell-like colony. (E) Morphology of established iPS cell line atpassage number 6 (clone 201B7). (F) Image of iPS cells with highmagnification. (G) Spontaneously differentiated cells in the center partof human iPS cell colonies. (H—N) Immunocytochemistry for SSEA-1 (H),SSEA-3 (I), SSEA-4 (J), TRA-1-60 (K), TRA-1-81 (L), TRA-2-4916E (M), andNanog (N). Nuclei were stained with Hoechst 33342 (blue). Bars=200 μm(B-E, G), 20 μm (F), and 100 μm (H—N).

FIGS. 33(A)-(C) show feeder dependency of human iPS cells. (A) Image ofiPS cells plated on gelatin-coated plate. (B) Images of iPS cellscultured on Matrigel-coated plates in MEF-conditioned medium (MEF-CCM).(C) Images of iPS cells cultured in ES medium on Matrigel-coated plateswith non-conditioned hES medium.

FIGS. 34(A)-(E) show expression of hES cell marker genes in human iPScells. (A) RT-PCR analysis of ES cell marker genes. (B) Western blotanalysis of ES cell marker genes. (C) Quantitative PCR for expression ofretroviral transgenes. The graph shows the average of three assays. Barsindicate standard deviation. (D) Bisulfite genomic sequencing of thepromoter regions of OCT3/4, REXJ and NANOG. Open and closed circlesindicate unmethylated and methylated CpGs. (E) Luciferase assays. Thegraphs show the average of the results from four assays. Bars indicatestandard deviation.

FIGS. 35(A)-(B) show high levels of telomerase activity and exponentialproliferation of human iPS cells. (A) Detection of telomerase activitiesby the TRAP method. Heat-inactivated (+) samples were used as negativecontrols. IC=internal control. (B) Growth curve of iPS cells. Shown areaverages and standard deviations in quadruplicate.

FIGS. 36(A)-(B) show genetic analyses of human iPS cells. (A) GenomicPCR revealed integration of all the four retroviruses in all clones. (B)Southern blot analyses with a c-MYC cDNA probe. Asterisk indicates theendogenous c-MYC alleles (2.7 kb). Arrowhead indicates mouse c-Mycalleles derived from SNL feeder cells (9.8 kb).

FIGS. 37(A)-(L) show embryoid body-mediated differentiation of human iPScells. (A) Floating culture of iPS cells at day 8. (B-E) Images ofdifferentiated cells at day 16 (B), neuron-like cells (C), epithelialcells (D), and cobblestone-like cells (E). (F-K) Immunocytochemistry ofalpha-fetoprotein (F), vimentin (G), α-smooth muscle actin (H), desmin(I), βIII-tubulin (J), and GFAP (K). Bars=200 μm (A, B) and 100 μm(C-K). Nuclei were stained with Hoechst 33342 (blue). (L) RT-PCRanalyses of various differentiation markers for the three germ layers.

FIGS. 38(A)-(E) show directed differentiations of human iPS cells. (A)Phase contrast image of differentiated iPS cells after 18 dayscultivation on PA6. (B) Immunocytochemistry of the cells shown in A withIII-tubulin (red) and tyrosine hydroxylase (green) antibodies. Nucleiwere stained with Hoechst 33342 (blue). (C) RT-PCR analyses ofdopaminergic neuron markers. (D) Phase contrast image of iPS cellsdifferentiated into cardiomyocytes. (E) RT-PCR analyses of cardiomyocytemarkers. Bars=200 μm (A, D) and 100 μm (B).

FIG. 39 shows hematoxylin and eosin staining of teratoma derived fromhuman iPS cells (clone 201B7).

FIG. 40 shows human iPS cells (phase contrast images) derived fromfibroblast-like synoviocytes (HFLS, clone 243H1) and BJ fibroblasts(clone 246G1). Bars=200 μm.

FIG. 41 shows expression of ES cell marker genes in iPS cells derivedfrom HFLS and BJ fibroblasts.

FIG. 42 shows embryoid body-mediated differentiation of iPS cellsderived from HFLS and BJ fibroblasts.

FIGS. 43(A)-(C) show the effect of family factors and the omission ofMyc on generation of iPS cells from Nanog-reporter MEFs. (A) Generationof iPS cells with family genes from MEF by Nanog selection. The numberof GFP-positive colonies is shown. The results of three independentexperiments were shown with different colors (white, gray, and black).The “4 factors” indicate the combination of Oct3/4, Sox2, Klf4, andc-Myc. (B) The effect of puromycin selection timing on iPS cellgeneration. Shown are GFP-positive colonies observed 28 days after thetransduction of the four factors or the three factors devoid of Myc. (C)The effect of puromycin selection timing on the percentage ofGFP-positive colonies per all colonies.

FIG. 44 shows teratomas derived from iPS cells, which were induced fromFbx15-reporter MEFs with family proteins.

FIG. 45 shows characterization of iPS cells induced from Nanog-reporterMEFs without Myc retroviruses. RT-PCR showing expression levels of EScell marker genes and the four factors. By using specific primer sets,total transcripts, transcripts from the endogenous genes (endo), and thetranscripts from the retroviruses (Tg) were distinguished for the fourfactors.

FIGS. 46(A)-(C) show generation of iPS cells without Myc retrovirusesfrom MEFs containing the Fbx15-reporter and the constitutively activeGFP-transgene. (A) Morphology of iPS cells generated without Mycretroviruses. The bar indicates 500 μm. (B) RT-PCR analyses of ES markergenes in ES, MEF, and iPS cells induced without Myc. (C) Chimerasderived from iPS cells induced without Myc (clones 142B-6 and -12).

FIGS. 47(A)-(D) show the efficient isolation of iPS cells without drugselection. (A) Morphology of iPS cells induced from adult TTF containingthe Nanog-GFP-IRES-Puro^(r) reporter. Cells were transduced with eitherthe four factors or the three factors devoid of Myc, together withDsRed, and then were cultured for 30 days without drug selection. Theexpression of the Nanog reporter (Nanog-GFP) and the DsRed retrovirus(Tg-DsRed) was examined by fluorescent microscopy. The bar indicates 500μm. (B) Morphology of iPS cells induced from adult TTF, which containeda DsRed transgene driven by a constitutively active promoter (ACTB,β-actin gene), but lacking the Nanog- or Fbx15-selection cassettes. Thecells were transduced with either the four factors or the three factorsdevoid of Myc, together with GFP, and then cultured for 30 days withoutdrug selection. The expression of the GFP retrovirus (Tg-GFP) wasexamined by fluorescent microscopy. The bar indicates 500 μm. (C) RT-PCRanalyses of ES maker genes in iPS cells generated from TTF without drugselection and ES cells. (D) Chimeras derived from iPS cells, which weregenerated from adult TTF without drug selection or the Myc retroviruses.

FIGS. 48(A)-(C) show induction of human iPS cells without Mycretroviruses. (A) The retroviruses for Oct3/4, Sox2 and Klf4 wereintroduced into BJ fibroblasts (246G) or HDF (253G). After 30 days, afew hES cell-like colonies emerged. These cells were expandable andshowed hES cell-like morphology. (B) The expression of ES cell markergenes in human iPS cells derived from HDF without Myc retroviruses(253G) or with Myc (253F). (C) Embryoid body-mediated differentiation ofhuman iPS cells generated without Myc retroviruses.

FIG. 49 shows results from experiments using six factors and twodifferent combinations of four factors. The vertical axis shows thenumber of colonies. The term “6F” refers to the six factors (klf4,c-myc, oct3/4, sox2, nanog and Lin-28), the term “Y4F” refers to thefirst combination of four factors (klf4, c-myc, oct3/4 and sox2), andthe term “T4F” refers to the second combination of four factors (oct3/4,sox2, nanog and Lin-28), respectively. The term “ES like” refers toES-like cell colony morphologically, and the term “total” shows totalnumber of ES-like cell colonies and non-ES like cell colonies. Exp#1,Exp#2, Exp#3, and Exp#4 show individual experimental results,respectively.

FIGS. 50(A)-(C) show a summary of data from experiments performed withmouse embryonic fibroblasts (MEFs). (A) 1.0×10⁵ MEF cells obtained fromNanog GFP^(tg/−)Fbx15^(−/−) mouse were seeded on gelatin coated 6 wellplates. Next day, four factors (Oct3/4, Klf4, Sox2, c-Myc) or threefactors (Oct3/4, Klf4, Sox2) were retrovirally transduced into thecells. After 4 days of the infection, cells were re-seeded 1 to 2 or 1to 6-ratio on 6 well plates covered with mitomycin C-treated STO cells.Drug selection was started at 14 days or 21 days. At day 28, GFPpositive cells were counted and cells were stained for alkalinephosphatase (AP) and crystal violet (CV). (B) Summary of the number ofthe GFP positive colonies from three independent experiments, Exp. #1,2, and 3. (C) Percentage of GFP positive colonies from three independentiPS experiments, Exp. #1, 2, and 3.

FIG. 51 shows a summary of data from experiments performed with adulthuman dermal fibroblasts. 1.0×10⁵ adult HDF cells expressing slc7a wereseeded on 6 well plates. Next day, four factors (Oct3/4, Klf4, Sox2,c-Myc) or three factors (Oct3/4, Klf4, Sox2) were retrovirallytransduced into the cells. After 6 days of the infection, 5.0×10⁵ cellswere re-seeded on 100 mm plates covered with 1.5×10⁶ of mitomycinC-treated STO cells. At day 7 the medium was replaced with Primate EScell medium supplemented with 4 ng/ml bFGF. This figure shows colonynumbers at 30 days after infections.

DETAILED DESCRIPTION OF THE INVENTION

Various investigations were conducted to address the aforementioned needfor pluripotent stem cells which can be derived from a patient's ownsomatic cells, and for nuclear reprogramming factors capable ofgenerating pluripotent stem cells from somatic cells. Investigationswere also conducted to identify nuclear reprogramming factors by usingthe screening method for a nuclear reprogramming factor disclosed inInternational Publication WO2005/80598. While International PublicationWO 2005/80598 discloses a screening method, this document fails todisclose any nuclear reprogramming factor. Furthermore, this documentfails to specify any nuclear reprogramming factor or candidate nuclearreprogramming factor which would be capable of generating an inducedpluripotent stem cell.

Ultimately, 24 kinds of candidate genes were found as genes relating tonuclear reprogramming, and among them, three kinds of the genes werefound as particularly preferred for nuclear reprogramming: sometimesthese genes are referred to as essential in certain embodiments. Asfurther discussed throughout the specification, the nuclearreprogramming factor of the present invention may contain one or morefactors relating to differentiation, development, proliferation or thelike and factors having other physiological activities, as well as othergene products which can function as a nuclear reprogramming factor. Thepresent invention was achieved on the basis of these findings.

The present invention provides at least the following advantages andfeatures: induced pluripotent stem (iPS) cells derived by nuclearreprogramming of a somatic cell, including methods for reprogramming ofa differentiated cell without using eggs, embryos, or embryonic stem(ES) cells.

As further discussed herein with respect to the general guidance for thereprogramming of differentiated cells and the examples, the presentinvention also provides various nuclear reprogramming factors capable ofgenerating pluripotent stem cells from somatic cells. The nuclearreprogramming factor may comprise one or more gene products. The nuclearreprogramming factor may also comprise a combination of gene products.Each nuclear reprogramming factor may be used alone or in combinationwith other nuclear reprogramming factors as disclosed herein. Inaddition, nuclear reprogramming may be performed with small molecules,compounds, or other agents such that iPS cells are obtained.

In a preferred embodiment, the nuclear reprogramming factor comprises agene product of each of the following three kinds of genes: an Octfamily gene, a Klf family gene, and a Sox family gene. According to amore preferred embodiment of the invention, there is provided theaforementioned factor comprising a gene product of each of the followingthree kinds of genes: Oct3/4, Klf4, and Sox2.

In another embodiment of the invention, there is provided a nuclearreprogramming factor comprising a gene product of each of the followingthree kinds of genes: an Oct family gene, a Klf family gene, and a Mycfamily gene. According to a preferred embodiment of the invention, thereis provided the aforementioned factor comprising a gene product of eachof the following three kinds of genes: Oct3/4, Klf4 and c-Myc.

According to another preferred embodiment, there is provided theaforementioned factor, which further comprises a gene product of thefollowing gene: a Sox family gene, and as a more preferred embodiment,there is provided the aforementioned factor, which comprises a geneproduct of Sox2.

According to still another preferred embodiment, there is provided theaforementioned factor, which comprises a cytokine together with the geneproduct of the Myc family gene, or alternatively, instead of the geneproduct of the Myc family gene. As a more preferred embodiment, there isprovided the aforementioned factor, wherein the cytokine is basicfibroblast growth factor (bFGF) and/or stem cell factor (SCF).Accordingly, it is understood that the nuclear reprogramming factor canbe with or without the Myc family gene.

According to particularly preferred embodiments, there is provided anuclear reprogramming factor for a somatic cell, which comprises a geneproduct of the TERT gene in addition to a gene product of each of an Octfamily gene, a Klf family gene, a Myc family gene, and a Sox familygene; and the aforementioned factor, which comprises a gene product orgene products of one or more kinds of genes selected from the groupconsisting of the following genes: SV40 Large T antigen (SEQ ID NO: 23),HPV16 E6 (SEQ ID NO: 24), HPV16 E7 (SEQ ID NO: 25), and Bmi1, inaddition to a gene product of each of the Oct family gene, the Klffamily gene, the Myc family gene, the Sox family gene, and the TERTgene.

In addition to these factors, there is provided the aforementionedfactor, which further comprises a gene product or gene products of oneor more kinds of genes selected from the group consisting of thefollowing: Fbx15, Nanog, ERas, ECAT15-2, Tcl1, and β-catenin.

There is also provided the aforementioned factor, which comprises a geneproduct or gene products of one or more kinds of genes selected from thegroup consisting of the following: ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3,Sox15, ECAT15-1, Fthl17, Sall4, Rex1, UTF1, Stella, Stat3, and Grb2.

The present invention also provides a nuclear reprogramming factorcomprising a gene product or gene products of one or more kinds of thefollowing genes: Oct3/4, Sox2, Klf4, Nanog, Lin-28, and c-Myc.

The present invention also provides a nuclear reprogramming factorcomprising any combination of gene products, small molecules and/orsubstances as described herein, further comprising one or more factorsimproving the efficiency of iPS cell induction, such as one or more geneproducts of a Sall1 or Sall4 gene.

In another aspect, the present invention provides a method for preparingan induced pluripotent stem cell by nuclear reprogramming of a somaticcell, which comprises the step of contacting the aforementioned nuclearreprogramming factor with the somatic cell.

There is also provided the aforementioned method, which comprises thestep of adding the aforementioned nuclear reprogramming factor to aculture of the somatic cell; the aforementioned method, which comprisesthe step of introducing a gene encoding the aforementioned nuclearreprogramming factor into the somatic cell; the aforementioned method,which comprises the step of introducing said gene into the somatic cellby using a recombinant vector containing at least one kind of geneencoding the aforementioned nuclear reprogramming factor; and theaforementioned method, wherein a somatic cell isolated from a patient isused as the somatic cell.

In another aspect, the present invention provides an induced pluripotentstem cell obtained by the aforementioned method. The present inventionalso provides a somatic cell derived by inducing differentiation of theaforementioned induced pluripotent stem cell.

The present invention further provides a method for stem cell therapy,which comprises the step of transplanting a somatic cell, wherein saidcell is obtained by inducing differentiation of an induced pluripotentstem cell obtained by the aforementioned method using a somatic cellisolated and collected from a patient, into said patient. Several kindsof, preferably approximately 200 kinds of iPS cells prepared fromsomatic cells derived from healthy humans can be stored in an iPS cellbank as a library of iPS cells, and one kind or more kinds of the iPScells in the library can be used for preparation of somatic cells,tissues, or organs that are free of rejection by a patient to besubjected to stem cell therapy.

The present invention further provides a method for evaluating aphysiological function or toxicity of a compound, a medicament, a poisonor the like by using various cells obtained by inducing differentiationof an induced pluripotent stem cell obtained by the aforementionedmethod.

The present invention also provides a method for improving ability ofdifferentiation and/or growth of a cell, which comprises the step ofcontacting the aforementioned nuclear reprogramming factor with thecell, and further provides a cell obtained by the aforementioned method,and a somatic cell derived by inducing differentiation of a cellobtained by the aforementioned method.

By using the nuclear reprogramming factor provided by the presentinvention, reprogramming of a differentiated cell nucleus can beconveniently and highly reproducibly induced without using embryos or EScells, and an induced pluripotent stem cell, as an undifferentiated cellhaving differentiation ability, pluripotency, and growth ability similarto those of ES cells, can be established. For example, an inducedpluripotent stem cell having high growth ability and differentiationpluripotency can be prepared from a patient's own somatic cell by usingthe nuclear reprogramming factor of the present invention. Cellsobtainable by differentiating said cell (for example, cardiac musclecells, insulin producing cells, nerve cells and the like) are extremelyuseful, because they can be utilized for stem cell transplantationtherapies for a variety of diseases such as cardiac insufficiency,insulin dependent diabetes mellitus, Parkinson's disease and spinal cordinjury, thereby the ethical problem concerning the use of human embryoand rejection after transplantation can be avoided. Further, variouscells obtainable by differentiating the induced pluripotent stem cell(for example, cardiac muscle cells, hepatic cells and the like) arehighly useful as systems for evaluating efficacy or toxicity ofcompounds, medicaments, poisons and the like.

As noted above, transplantation of ES cells has a problem of causingrejection in the same manner as organ transplantation. Moreover, from anethical viewpoint, there are many dissenting opinions against the use ofES cells, which are established by destroying human embryos.

The present invention provides at least the following advantages andfeatures:

Identification of Nuclear Reprogramming Factors

As will be further disclosed below, the nuclear reprogramming factor ofthe present invention may contain one or more factors relating todifferentiation, development, proliferation or the like and factorshaving other physiological activities, as well as other gene productswhich can function as a nuclear reprogramming factor. It is understoodthat such embodiments fall within the scope of the present invention,and the present invention is, in other words, directed to factorsinducing pluripotent stem cells and various methods of obtaining inducedpluripotent stem cells, including various manners of reprogrammingdifferentiated cells as well as various manners of culturing,maintaining, and differentiating the induced pluripotent stem cells.

Furthermore, by using somatic cells in which only one or two genes amongthe three kinds of genes Oct3/4, Klf4, and c-Myc are expressed, othergene products which can function as a nuclear reprogramming factor canbe identified by, for example, performing screening for a gene productwhich can induce nuclear reprogramming of said cells. For example,depending on the kinds of genes expressed in a differentiated cell, oneor more genes useful as a reprogramming factor can be determined usingthe guidance herein provided. According to the present invention, theaforementioned screening method is also provided as a novel method forscreening for a nuclear reprogramming factor. In other words, thepresent invention is not limited to any particular combination ofnuclear reprogramming factors and the nuclear reprogramming factors ofthe present invention can be identified by screening methods, forexample, the aforementioned screening method.

In one embodiment, the nuclear reprogramming factor of the presentinvention is characterized in that it comprises one or more geneproducts. As a means for confirming the nuclear reprogramming factor ofthe present invention, for example, the screening method for nuclearreprogramming factors disclosed in International Publication WO2005/80598 can be used. The entire disclosure of the aforementionedpublication is incorporated into the disclosure of the specification byreference. By referring to the aforementioned publication, those skilledin the art can perform screening of nuclear reprogramming factors toconfirm the existence and the action of the reprogramming factor of thepresent invention.

For example, as an experimental system allowing for observation of thereprogramming phenomenon, a mouse can be used in which the βgeo (afusion gene of the β galactosidase gene and the neomycin resistancegene) is knocked into the Fbx15 locus (Tokuzawa et al., Mol. Cell. Biol.23:2699-708, 2003). The details are described in the examples of thespecification. The mouse Fbx15 gene is a gene specifically expressed indifferentiation pluripotent cells such as ES cells and early embryos. Ina homomutant mouse in which βgeo is knocked into the mouse Fbx15 gene soas to be deficient in the Fbx15 function, abnormal phenotypes includingthose relating to differentiation pluripotency or generation are notgenerally observed. In this mouse, the expression of the βgeo iscontrolled by the enhancer and promoter of the Fbx15 gene, anddifferentiated somatic cells in which βgeo is not expressed havesensitivity to G418. In contrast, βgeo knockin homomutant ES cells haveresistance against G418 at an extremely high concentration (higher than12 mg/ml). By utilizing this phenomenon, an experimental system can beconstructed to visualize reprogramming of somatic cells.

By applying the aforementioned experimental system, fibroblasts (Fbx15^(βgeo/βgeo) MEFs) can be first isolated from an embryo of the βgeoknockin homomutant mouse (13.5 days after fertilization). The MEFs donot express the Fbx15 gene, and accordingly also do not express βgeo togive sensitivity to G418. However, when the MEFs are fused with geneticmanipulation-free ES cells (also have sensitivity to G418), βgeo isexpressed and the cells become G418-resistant as a result ofreprogramming of nuclei of MEFs. Therefore, by utilizing thisexperimental system, the reprogramming phenomenon can be visualized asG418 resistance.

Nuclear reprogramming factors can be selected by using theaforementioned experimental system. As candidates of genes relevant tonuclear reprogramming factors, a plurality of genes can be selectedwhich show specific expression in ES cells or of which important rolesin the maintenance of pluripotency of ES cells are suggested, and it canbe confirmed whether or not each candidate gene can induce nuclearreprogramming alone or in an appropriate combination thereof. Forexample, a combination of all of the selected primary candidate genes isconfirmed to be capable of inducing the reprogramming of differentiatedcells into a state close to that of ES cells. Combinations are thenprepared by withdrawing each individual gene from the aforementionedcombination, and the same actions of the combinations are confirmed inorder to select each secondary candidate gene whose absence causes areduction of the reprogramming induction ability or loss of thereprogramming induction ability. By repeating similar steps for thesecondary candidate genes selected as described above, an essentialcombination of nuclear reprogramming genes can be selected, and it canbe confirmed that a combination of gene products of each of the threekinds of genes, e.g., an Oct family gene, a Klf family gene, and a Mycfamily gene, acts as a nuclear reprogramming factor. It can be furtherconfirmed that a combination of a gene product of a Sox family geneadditionally with the gene products of the aforementioned three kinds ofgenes has extremely superior characteristics as a nuclear reprogrammingfactor. Specific examples of the selection method for the nuclearreprogramming factors are demonstrated in the examples of thespecification.

Therefore, by referring to the above general explanations and specificexplanations of the examples, those skilled in the art can readilyconfirm that the combination of these three kinds of genes induces thereprogramming of somatic cells, and that the combination of these threekinds of gene products is essential for nuclear reprogramming in certainembodiments. Thus, the embodiments herein illustrate variouscombinations of gene products and/or nuclear reprogramming factors whichcan provide iPS cells. In other words, based on the disclosure providedherein, one of ordinary skill in the art would know from the disclosedexamples and/or readily determine which combination and/or combinationsof nuclear reprogramming factors, including gene products, can generatepluripotent stem cells.

Nuclear Reprogramming Factor (NRF)

In a preferred embodiment, the NRF comprises a gene product. The nuclearreprogramming factor can be used to induce reprogramming of adifferentiated cell without using eggs, embryos, or ES cells, toconveniently and highly reproducibly establish an induced pluripotentstem cell having pluripotency and growth ability similar to those of EScells. For example, the nuclear reprogramming factor can be introducedinto a cell by transducing the cell with a recombinant vector comprisinga gene encoding the nuclear reprogramming factor. Accordingly, the cellcan express the nuclear reprogramming factor expressed as a product of agene contained in the recombinant vector, thereby inducing reprogrammingof a differentiated cell.

The nuclear reprogramming factor may comprise a protein or peptide. Theprotein may be produced from the aforementioned gene, or alternatively,in the form of a fusion gene product of said protein with anotherprotein, peptide or the like. The protein or peptide may be afluorescent protein and/or a fusion protein. For example, a fusionprotein with green fluorescence protein (GFP) or a fusion gene productwith a peptide such as a histidine tag can also be used. Further, bypreparing and using a fusion protein with the TAT peptide derived formthe virus HIV, intracellular uptake of the nuclear reprogramming factorthrough cell membranes can be promoted, thereby enabling induction ofreprogramming only by adding the fusion protein to a medium thusavoiding complicated operations such as gene transduction. Sincepreparation methods of such fusion gene products are well known to thoseskilled in the art, skilled artisans can easily design and prepare anappropriate fusion gene product depending on the purpose.

Nuclear reprogramming may also be accomplished with one or more smallmolecules, compounds, including inorganic and organic compounds, ormixtures thereof, extracts, epigenetic factors, and/or other componentsof the cytoplasm of a pluripotent cell.

In a particularly preferred embodiment, the nuclear reprogramming factormay comprise one or more gene products of each of the following threekinds of genes: an Oct family gene, a Klf family gene, and a Sox familygene.

In another preferred embodiment, the nuclear reprogramming factor maycomprise one or more gene products of each of: an Oct family gene, a Klffamily gene, and a Myc family gene.

The nuclear reprogramming factor may also comprise one or more geneproducts of each of: an Oct family gene, a Klf family gene, a Myc familygene, and a Sox family gene.

The nuclear reprogramming factor may also comprise one or more geneproducts of each of: an Oct family gene, a Klf family gene, and acytokine. In one exemplary embodiment, the above-referenced nuclearreprogramming factor may further comprise one or more gene products of aMyc family gene. In another exemplary embodiment, the above referencednuclear reprogramming factor may further comprise one or more geneproducts of a Sox family gene.

The cytokines of the present invention are not particularly limited. Forexample, the cytokine may comprise basic fibroblast growth factor(bFGF/FGF2) or stem cell factor (SCF).

With regard to gene family members, the nuclear reprogramming factor maycomprise any combination of members from one or more gene families. Forexample, a combination of one or more gene products of Oct3/4, Klf4, andc-Myc. Examples of the Oct family gene include, for example, Oct3/4,Oct1A, Oct6, and the like. Oct3/4 is a transcription factor belonging tothe POU family, and is reported as a marker of undifferentiated cells(Okamoto et al., Cell 60:461-72, 1990). Oct3/4 is also reported toparticipate in the maintenance of pluripotency (Nichols et al., Cell95:379-91, 1998). Examples of the Klf family gene include Klf1, Klf2,Klf4, Klf5 and the like. Klf4 (Kruppel like factor-4) is reported as atumor repressing factor (Ghaleb et al., Cell Res. 15:92-96, 2005).Examples of the Myc family gene include c-Myc, N-Myc, L-Myc and thelike. c-Myc is a transcription control factor involved indifferentiation and proliferation of cells (Adhikary & Eilers, Nat. Rev.Mol. Cell. Biol. 6:635-45, 2005), and is also reported to be involved inthe maintenance of pluripotency (Cartwright et al., Development132:885-96, 2005). The NCBI accession numbers of the genes of thefamilies other than Oct3/4, Klf4 and c-Myc are set in TABLE 1 asfollows:

TABLE 1 Mouse Human Klf1 Kruppel-like factor 1 NM_010635 NM_006563(erythroid) Klf2 Kruppel-like factor 2 NM_008452 NM_016270 (lung) Klf5Kruppel-like factor 5 NM_009769 NM_001730 c-Myc myelocytomatosisoncogene NM_010849 NM_002467 N-Myc v-Myc myelocytomatosis viralNM_008709 NM_005378 related oncogene, neuroblastoma derived (avian)L-Myc v-Myc myelocytomatosis viral NM_008506 NM_005376 oncogene homolog1, lung carcinoma derived (avian) Oct1A POU domain, class 2, NM_198934NM_002697 transcription factor 1 Oct6 POU domain, class 3, NM_011141NM_002699 transcription factor 1

All of these genes are those commonly existing in mammals includinghuman, and for use of the aforementioned gene products in the presentinvention, genes derived from arbitrary mammals (those derived frommammals such as mouse, rat, bovine, ovine, horse, and ape) can be used.In addition to wild-type gene products, mutant gene products includingsubstitution, insertion, and/or deletion of several (for example, 1 to10, preferably 1 to 6, more preferably 1 to 4, still more preferably 1to 3, and most preferably 1 or 2) amino acids and having similarfunction to that of the wild-type gene products can also be used. Forexample, as a gene product of c-Myc, a stable type product (T58A) may beused as well as the wild-type product. The above explanation may beapplied similarly to the other gene products.

The nuclear reprogramming factor of the present invention may comprise agene product other than the aforementioned three kinds of gene products.An example of such gene product includes a gene product of a Sox familygene. Examples of the Sox family genes include, for example, Sox1, Sox3,Sox7, Sox15, Sox17 and Sox18, and a preferred example includes Sox2. Anuclear reprogramming factor comprising at least a combination of thegene products of four kinds of genes, an Oct family gene (for example,Oct3/4), a Klf family gene (for example, Klf4), a Myc family gene (forexample, c-Myc), and a Sox family gene (for example, Sox2) is apreferred embodiment of the present invention from a viewpoint ofreprogramming efficiency, and in particular, a combination of a geneproduct of a Sox family gene is sometimes preferred to obtainpluripotency. Sox2, expressed in an early development process, is a geneencoding a transcription factor (Avilion et al., Genes Dev. 17:126-40,2003). The NCBI accession numbers of Sox family genes other than Sox2are in TABLE 2 as follows.

TABLE 2 Mouse Human Sox1 SRY-box containing gene 1 NM_009233 NM_005986Sox3 SRY-box containing gene 3 NM_009237 NM_005634 Sox7 SRY-boxcontaining gene 7 NM_011446 NM_031439 Sox15 SRY-box containing gene 15NM_009235 NM_006942 Sox17 SRY-box containing gene 17 NM_011441 NM_022454Sox18 SRY-box containing gene 18 NM_009236 NM_018419

Further, a gene product of a Myc family gene may be replaced with acytokine. As the cytokine, for example, SCF, bFGF or the like ispreferred. However, cytokines are not limited to these examples.

As a more preferred embodiment, an example includes a factor whichinduces immortalization of cells, in addition to the aforementionedthree kinds of gene products, preferably, the four kinds of geneproducts. For example, an example includes a combination of a factorcomprising a gene product of the TERT gene. In another exemplaryembodiment, the nuclear reprogramming factor comprises any of theaforementioned gene products in combination with a factor comprising agene product or gene products of one or more kinds of the followinggenes: SV40 Large T antigen, HPV16 E6, HPV16 E7, and Bmi1. TERT isessential for the maintenance of the telomere structure at the end ofchromosome at the time of DNA replication, and the gene is expressed instem cells or tumor cells in humans, whilst it is not expressed in manysomatic cells (Horikawa et al., P.N.A.S. USA 102:18437-442, 2005). SV40Large T antigen, HPV16 E6, HPV16 E7, or Bmi1 was reported to induceimmortalization of human somatic cells in combination with Large Tantigen (Akimov et al., Stem Cells 23:1423-33, 2005; Salmon et al., Mol.Ther. 2:404-14, 2000). These factors are extremely useful particularlywhen iPS cells are induced from human cells. The NCBI accession numbersof TERT and Bmi1 genes are listed in TABLE 3 as follows.

TABLE 3 Mouse Human TERT telomerase reverse NM_009354 NM_198253transcriptase Bmi1 B lymphoma Mo-MLV NM_007552 NM_005180 insertionregion 1

Furthermore, a gene product or gene products of one or more kinds ofgenes selected from the group consisting of the following: Fbx15, Nanog,ERas, ECAT15-2, Tcl1, and β-catenin may be combined. As a particularlypreferred embodiment from a viewpoint of reprogramming efficiency, anexample includes a nuclear reprogramming factor comprising a total often kinds of gene products, wherein gene products of Fbx15, Nanog, ERas,ECAT15-2, Tcl1, and β-catenin are combined with the aforementioned fourkinds of gene products. Fbx15 (Tokuzawa et al., Mol. Cell. Biol.23:2699-708, 2003), Nanog (Mitsui et al., Cell 113:631-42, 2003), ERas(Takahashi et al. Nature 423:541-45, 2003), and ECAT15-2 (Bortvin etal., Development 130:1673-80, 2003) are genes specifically expressed inES cells. Tcl1 is involved in the activation of Akt (Bortvin et al.,Development 130:1673-80, 2003), and β-catenin is an important factorconstituting the Wnt signal transmission pathway, and also reported tobe involved in the maintenance of pluripotency (Sato et al, Nat. Med.10:55-63, 2004).

Further, the nuclear reprogramming factor of the present invention maycomprise, for example, a gene product or gene products of one or morekinds of genes selected from the group consisting of the following:ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fthl17, Sall4, Rex1,UTF1, Stella, Stat3, and Grb2. ECAT1, Esg1, ECAT8, Gdf3, and ECAT15-1are genes specifically expressed in ES cells (Mitsui et al., Cell113:631-42, 2003). Dnmt3L is a DNA methylating enzyme-related factor,and Sox 15 is a class of genes expressed in an early development processand encoding transcription factors (Maruyama et al., J. Biol. Chem.280:24371-79, 2005). Fthl17 encodes ferritin heavy polypeptide-like 17(colLoriot et al., Int. J. Cancer 105:371-76, 2003), Sall4 encodes a Znfinger protein abundantly expressed in embryonic stem cells (Kohlhase etal., Cytogenet. Genome Res. 98:274-77, 2002), and Rex1 encodes atranscription factor locating downstream from Oct3/4 (Ben-Shushan etal., Mol. Cell. Biol. 18:1866-78, 1998). UTF1 is a transcriptioncofactor locating downstream from Oct3/4, and it is reported that thesuppression of the proliferation of ES cells is induced when this factoris suppressed (Okuda et al., EMBO J. 17:2019-32, 1998). Stat3 is asignal factor for proliferation and differentiation of cells. Theactivation of Stat3 triggers the operation of LIF, and thereby thefactor plays an important role for the maintenance of pluripotency (Niwaet al., Genes Dev. 12:2048-60, 1998). Grb2 encodes a protein mediatingbetween various growth factor receptors existing in cell membranes andthe Ras/MAPK cascade (Cheng et al. Cell 95:793-803, 1998).

However, as noted above, the gene products which may be included in thenuclear reprogramming factor of the present invention are not limited tothe gene products of the genes specifically explained above. The nuclearreprogramming factor of the present invention may contain one or morefactors relating to differentiation, development, proliferation or thelike and factors having other physiological activities, as well as othergene products which can function as a nuclear reprogramming factor. Itis understood that such embodiments fall within the scope of the presentinvention. By using somatic cells in which only one or two genes amongthe three kinds of the gene Oct3/4, Klf4, and c-Myc are expressed, theother gene products which can function as a nuclear reprogramming factorcan be identified by, for example, performing screening for a geneproduct which can induce nuclear reprogramming of said cells. Accordingto the present invention, the aforementioned screening method is alsoprovided as a novel method for screening for a nuclear reprogrammingfactor.

Cells of the Invention and Methods of Generating the Same

By using the nuclear reprogramming factor of the present invention, thenucleus of a somatic cell can be reprogrammed to obtain an inducedpluripotent stem cell. In the specification, the term “inducedpluripotent stem cells” means cells having properties similar to thoseof ES cells, and more specifically, the term encompassesundifferentiated cells having pluripotency and growth ability. However,the term should not be construed narrowly in any sense, and should beconstrued in the broadest sense. The method for preparing inducedpluripotent stem cells by using a nuclear reprogramming factor isexplained in International Publication WO2005/80598 (the term “ES-likecells” is used in the publication), and a means for isolating inducedpluripotent stem cells is also specifically explained. Therefore, byreferring to the aforementioned publication, those skilled in the artcan easily prepare induced pluripotent stem cells by using the nuclearreprogramming factor of the present invention. Methods for preparinginduced pluripotent stem cells from somatic cells by using the nuclearreprogramming factor of the present invention are not particularlylimited. Any method may be employed as long as the nuclear reprogrammingfactor can contact with somatic cells under an environment in which thesomatic cells and induced pluripotent stem cells can proliferate. Anadvantage of the present invention is that an induced pluripotent stemcell can be prepared by contacting a nuclear reprogramming factor with asomatic cell in the absence of eggs, embryos, or embryonic stem (ES)cells.

For example, a gene product contained in the nuclear reprogrammingfactor of the present invention may be added to a medium. Alternatively,by using a vector containing a gene that is capable of expressing thenuclear reprogramming factor of the present invention, a means oftransducing said gene into a somatic cell may be employed. When suchvector is used, two or more kinds of genes may be incorporated into thevector, and each of the gene products may be simultaneously expressed ina somatic cell. When one or more of the gene products contained in thenuclear reprogramming factor of the present invention are alreadyexpressed in a somatic cell to be reprogrammed, said gene products maybe excluded from the nuclear reprogramming factor of the presentinvention. It is understood that such embodiments fall within the scopeof the present invention.

As indicated above, the nuclear reprogramming factor of the presentinvention can be used to generate iPS cells from differentiated adultsomatic cells. In the preparation of induced pluripotent stem cells byusing the nuclear reprogramming factor of the present invention, typesof somatic cells to be reprogrammed are not particularly limited, andany kind of somatic cells may be used. For example, matured somaticcells may be used, as well as somatic cells of an embryonic period.Other examples of cells capable of being generated into iPS cells and/orencompassed by the present invention include mammalian cells such asfibroblasts, B cells, T cells, dendritic cells, ketatinocytes, adiposecells, epithelial cells, epidermal cells, chondrocytes, cumulus cells,neural cells, glial cells, astrocytes, cardiac cells, esophageal cells,muscle cells, melanocytes, hematopoietic cells, pancreatic cells,hepatocytes, macrophages, monocytes, mononuclear cells, and gastriccells, including gastric epithelial cells. The cells can be embryonic,or adult somatic cells, differentiated cells, cells with an intactnuclear membrane, non-dividing cells, quiescent cells, terminallydifferentiated primary cells, and the like.

Induced pluripotent stem cells may express any number of pluripotentcell markers, including: alkaline phosphatase (AP); ABCG2; stagespecific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60;TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; βIII-tubulin; α-smoothmuscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1;zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cellassociated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7;ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sall4;undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53;G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a;Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4;Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3;CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-celllymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; other general markersfor pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3;Grb2; SV40 Large T Antigen; HPV16 E6; HPV16 E7, β-catenin, and Bmi1.Such cells can also be characterized by the down-regulation of markerscharacteristic of the differentiated cell from which the iPS cell isinduced. For example, iPS cells derived from fibroblasts may becharacterized by down-regulation of the fibroblast cell marker Thy1and/or up-regulation of SSEA-1. It is understood that the presentinvention is not limited to those markers listed herein, and encompassesmarkers such as cell surface markers, antigens, and other gene productsincluding ESTs, RNA (including microRNAs and antisense RNA), DNA(including genes and cDNAs), and portions thereof.

When induced pluripotent stem cells are used for therapeutic treatmentof diseases, it is desirable to use somatic cells isolated frompatients. For example, somatic cells involved in diseases, somatic cellsparticipating in therapeutic treatment of diseases and the like can beused. A method for selecting induced pluripotent stem cells that appearin a medium according to the method of the present invention is notparticularly limited, and a well-known means may be suitably employed,for example, a drug resistance gene or the like can be used as a markergene to isolate induced pluripotent stem cells using drug resistance asan index. Various media that can maintain undifferentiated state andpluripotency of ES cells and various media which cannot maintain suchproperties are known in this field, and induced pluripotent stem cellscan be efficiently isolated by using a combination of appropriate media.Differentiation and proliferation abilities of isolated inducedpluripotent stem cells can be easily confirmed by those skilled in theart by using confirmation means widely applied to ES cells.

Thus, another preferred embodiment of the invention comprises apluripotent stem cell induced by reprogramming a somatic cell in theabsence of eggs, embryos, or embryonic stem (ES) cells. The pluripotentstem cell can be a mammalian cell, for example a mouse, human, rat,bovine, ovine, horse, hamster, dog, guinea pig, or ape cell. Forexample, direct reprogramming of somatic cells provides an opportunityto generate patient- or disease-specific pluripotent stem cells. MouseiPS cells are indistinguishable from ES cells in morphology,proliferation, gene expression, and teratoma formation. Furthermore,when transplanted into blastocysts, mouse iPS cells can give rise toadult chimeras, which are competent for germline transmission (Maheraliet al., Cell Stem Cell 1:55-70, 2007; Okita et al., Nature 448:313-17,2007; Wemig et al., Nature 448:318-324, 2007). Human iPS cells are alsoexpandable and indistinguishable from human embryonic stem (ES) cells inmorphology and proliferation. Furthermore, these cells can differentiateinto cell types of the three germ layers in vitro and in teratomas.

The present invention also provides for the generation of somatic cellsderived by inducing differentiation of the aforementioned pluripotentstem cells. The present invention thus provides a somatic cell derivedby inducing differentiation of the aforementioned induced pluripotentstem cell.

In another embodiment, there is disclosed a method for improvingdifferentiation ability and/or growth ability of a cell, which comprisescontacting a nuclear reprogramming factor with a cell.

In a particularly preferred embodiment, the present invention comprisesa method for stem cell therapy comprising: (1) isolating and collectinga somatic cell from a patient; (2) inducing said somatic cell from thepatient into an iPS cell; (3) inducing differentiation of said iPS cell,and (4) transplanting the differentiated cell from step (3) into thepatient.

In another preferred embodiment, the present invention includes a methodfor evaluating a physiological function of a compound comprisingtreating cells obtained by inducing differentiation of an inducedpluripotent stem cell with the compound.

A method for evaluating the toxicity of a compound comprising treatingcells obtained by inducing differentiation of an induced pluripotentstem cell in the presence of the compound.

EXAMPLES

Various terms, abbreviations and designations for the raw materials andtests used in the following Examples are explained as follows:

Abbreviations

iPS cell (induced pluripotent stem cell)

NRF (nuclear reprogramming factor)

ES cell (embryonic stem cell)

TTF (tail tip fibroblast)

MEF (mouse embryonic fibroblast)

HDF (human dermal fibroblast)

bFGF (basic fibroblast growth factor)

SCF (stem cell factor)

GFP (green fluorescent protein)

The present invention will be more specifically explained with referenceto examples. However, the scope of the present invention is not limitedto the following examples.

Example 1 Selection of A Nuclear Reprogramming Factor

In order to identify reprogramming factors, an experimental system foreasy observation of the reprogramming phenomenon is required. As anexperimental system, a mouse in which βgeo (a fusion gene ofβ-galactosidase gene and neomycin resistance gene) was knocked into theFbx15 locus (Tokuzawa et al., Mol. Cell. Biol. 23:2699-708, 2003) wasused. The mouse Fbx15 gene is a gene specifically expressed indifferentiation pluripotent cells such as ES cells and early embryos.However, in a homomutant mouse in which βgeo was knocked into the mouseFbx15 gene so as to delete the function of Fbx15, no abnormal phenotypesincluding those concerning differentiation pluripotency or developmentwere observed. In this mouse, expression control of βgeo is attained bythe enhancer and promoter of the Fbx15 gene. Specifically, βgeo is notexpressed in differentiated somatic cells, and they have sensitivity toG418. In contrast, the βgeo knockin homomutant ES cells have resistanceagainst G418 at an extremely high concentration (higher than 12 mg/ml).By utilizing the above phenomenon, an experimental system forvisualizing the reprogramming of somatic cells was constructed.

In the aforementioned experimental system, fibroblasts(Fbx15^(βgeo/βgeo), MEFs) were first isolated from an embryo of the βgeoknockin homomutant mouse (13.5 days after fertilization). Since MEFs donot express the Fbx15 gene, the cells also do not express βgeo and thushave sensitivity to G418. Whilst when the MEFs are fused with ES cellsthat have not been gene-manipulated (also having sensitivity to G418),the nuclei of MEFs are reprogrammed, and as a result, βgeo is expressedto give G418-resistance. The reprogramming phenomenon can thus bevisualized as G418 resistance by using this experimental system(International Publication WO2005/80598). Searches for reprogrammingfactors were performed by using the aforementioned experimental system(FIG. 1), and total 24 kinds of genes were selected as candidatereprogramming factors, including genes showing specific expression in EScells and genes suggested to have important roles in the maintenance ofdifferentiation pluripotency of ES cells. These genes are shown inTABLES 4 and 5 below. For β-catenin (#21) and c-Myc (#22), active typemutants (catenin: S33Y, c-Myc: T58A) were used.

TABLE 4 Number Name of Gene Explanation of Gene 1 ECAT1 ES cellassociated transcript 1 (ECAT1) 2 ECAT2 developmental pluripotencyassociated 5 (DPPA5), ES cell specific gene 1 (ESG1) 3 ECAT3 F-boxprotein 15 (Fbx15), 4 ECAT4 homeobox transcription factor Nanog 5 ECAT5ES cell expressed Ras (ERas) 6 ECAT7 DNA (cytosine-5-)-methyltransferase3-like (Dnmt31), valiant 1 7 ECAT8 ES cell associated transcript 8(ECAT8) 8 ECAT9 growth differentiation factor 3 (Gdf3) 9 ECAT10 SRY-boxcontaining gene 15 (Sox15) 10 ECAT15-1 developmental pluripotencyassociated 4 (Dppa4), variant 1 11 ECAT15-2 developmental pluripotencyassociated 2 (Dppa2) 12 Fthl17 ferritin, heavy polypeptide-like 17(Fthl17) 13 Sall4 sal-like 4 (Drosophila) (Sall4), transcript variant a14 Oct3/4 POU domain, class 5, transcription factor 1 (Pou5f1) 15 Sox2SRY-box containing gene 2 (Sox2) 16 Rex1 zinc finger protein 42 (Zfp42)17 Utf1 undifferentiated embryonic cell transcription factor 1 (Utf1) 18Tcl1 T-cell lymphoma breakpoint 1 (Tcl1) 19 Stella developmentalpluripotency-associated 3 (Dppa3) 20 Klf4 Kruppel-like factor 4 (gut)(Klf4) 21 β-catenin catenin (cadherin associated protein), beta 1, 88kDa (Ctnnb1) 22 c-Myc myelocytomatosis oncogene (Myc) 23 Stat3 signaltransducer and activator of transcription 3 (Stat3), transcript variant1 24 Grb2 growth factor receptor bound protein 2 (Grb2)

TABLE 5 Name of NCBI accession number Number Gene Characteristic FeatureMouse Human 1 ECAT1 Gene specifically expressed in ES AB211060 AB211062cell 2 ECAT2 Gene specifically expressed in ES NM_025274 NM_001025290cell 3 ECAT3 Gene specifically expressed in ES NM_015798 NM_152676 cell4 ECAT4 Transcription factor having AB093574 NM_024865 homeodomain,essential factor for differentiation pluripotency maintenance 5 ECAT5Ras family protein, ES cell growth NM_181548 NM_181532 promoting factor6 ECAT7 DNA methylation enzyme-related NM_019448 NM_013369 factor,essential for imprinting 7 ECAT8 Gene specifically expressed in ESAB211061 AB211063 cell, having Tudor domain 8 ECAT9 Gene specificallyexpressed in ES NM_008108 NM_020634 cell, belonging to TGFβ family 9ECAT10 Gene specifically expressed in ES NM_009235 NM_006942 cell, SRYfamily transcription factor 10 ECAT15-1 Gene specifically expressed inES NM_028610 NM_018189 cell 11 ECAT15-2 Gene specifically expressed inES NM_028615 NM_138815 cell 12 Fthl17 Gene specifically expressed in ESNM_031261 NM_031894 cell, similar to ferritin heavy chain 13 Sall4 Genespecifically expressed in ES NM_175303 NM_020436 cell, Zn finger protein14 Oct3/4 POU family transcription factor, NM_013633 NM_002701 essentialfor pluripotency maintenance 15 Sox2 SRY family transcription factor,NM_011443 NM_003106 essential for pluripotency maintenance 16 Rex1 Genespecifically expressed in ES NM_009556 NM_174900 cell, Zn finger protein17 Utf1 Transcription regulation factor NM_009482 NM_003577 highlyexpressed in ES cell, promoting growth of ES 18 Tcl1 Oncogene activatingAKT, NM_009337 NM_021966 abundantly expressed in ES cell 19 Stella Genespecifically expressed in ES NM_139218 NM_199286 cell 20 Klf4 Abundantlyexpressed in ES cell, NM_010637 NM_004235 both actions as antioncogeneand oncogene are reported 21 β-catenin Transcription factor activated byNM_007614 NM_001904 Wnt signal, involvement in pluripotency maintenanceis reported 22 c-Myc Transcription control factor NM_010849 NM_002467participating in cell proliferation and differentiation and oncogene,involvement in pluripotency maintenance is reported 23 Stat3Transcription factor activated by LIF NM_213659 NM_139276 signal,considered essential for pluripotency maintenance of mouse ES cells 24Grb2 Adapter protein mediating growth NM_008163 NM_002086 factorreceptors and Ras/MAPK cascade

cDNAs of these genes were inserted into the retroviral vector pMX-gw bythe Gateway technology. First, each of the 24 genes was infected intoFbx15^(βgeo/βgeo) MEFs, and then G418 selection was performed under EScell culture conditions. However, no G418-resistant colony was obtained.Next, the retroviral vectors of all of the 24 genes were simultaneouslyinfected into Fbx15^(βgeo/βgeo) MEFs. When G418 selection was performedunder ES cell culture conditions, a plurality of drug resistant colonieswere obtained. These colonies were isolated, and cultivation wascontinued. It was found that cultivation of these cells over a longperiod of time could be performed, and that these cells had morphologysimilar to that of ES cells (FIG. 2). In the figure, iPS cells representinduced pluripotent stem cells (also called “ES like cells”, “ES-likecells”, or “ESL cells”), ES represents embryonic stem cells, and MEFrepresents differentiated cells (embryonic fibroblasts).

When expression profiles of the marker genes were examined by RT-PCR,undifferentiation markers such as Nanog and Oct3/4 were found to beexpressed (FIG. 3). It was found that the expression of Nanog was closeto that of ES cells, whereas the expression of Oct3/4 was lower thanthat of ES cells. When DNA methylation status was examined by thebisulfite genomic sequencing, it was found that the Nanog gene and Fbx15gene were highly methylated in MEFs, whereas they were demethylated inthe iPS cells (FIG. 4). About 50% of IGF2 gene, an imprinting gene, wasmethylated both in the MEF and iPS cells. Since it was known that theimprinting memory was deleted and the IGF2 gene was almost completelydemethylated in the primordial germ cells at 13.5 days afterfertilization, from which the Fbx15^(βgeo/βgeo) MEFs were isolated, itwas concluded that iPS cells were not derived from primordial germ cellscontaminated in the Fbx15^(βgeo/βgeo) MEFs. The above resultsdemonstrated that reprogramming of the differentiated cells (MEFs) intoa state close to that of ES cells was able to be induced with thecombination of the 24 kinds of factors.

Then, studies were made as to whether or not all of the 24 kinds ofgenes were required for the reprogramming. With withdrawal of eachindividual gene, 23 genes were transfected into the Fbx15^(βgeo/βgeo)MEFs. As a result, for 10 genes, colony formation was found to beinhibited with each withdrawal thereof (FIG. 5, the gene numberscorrespond to the gene numbers shown in TABLE 4, and the genes are thefollowing 10 kinds of genes: #3, #4, #5, #11, #14, #15, #18, #20, #21,and #22). When these ten genes were simultaneously transfected into theFbx15^(βgeo/βgeo) MEFs, G418-resistant colonies were significantly moreefficiently obtained as compared to simultaneous transfection with the24 genes.

Furthermore, 9 genes, with drawal of each individual gene from the 10genes, were transfected into Fbx15^(βgeo/βgeo) MEFs. As a result, it wasfound that G418-resistant iPS cell colonies were not formed when each of4 kinds of genes (# 14, # 15, #20, or #22) was withdrawn (FIG. 6).Therefore, it was suggested that these four kinds of genes, among theten genes, had particularly important roles in the induction ofreprogramming.

Example 2 Induction of Reprogramming with a Combination of 4 Kinds ofGenes

It was examined whether or not induction of reprogramming of somaticcells was achievable with the four kinds of genes of which particularimportance was suggested among the 10 genes. By using the combination ofthe aforementioned 10 kinds of genes, the combination of theaforementioned 4 kinds of genes, combinations of only 3 kinds of genesamong the 4 kinds of genes, and combinations of only 2 kinds of genesamong the 4 kinds of genes, these sets of genes were retrovirallytransduced into the MEF cells as somatic cells in which βgeo was knockedinto the Fbx15 gene. As a result, when the 4 kinds of genes weretransduced, 160 G418-resistant colonies were obtained. Although thisresult was almost the same as that obtained by the transduction with the10 kinds of genes (179 colonies), the colonies obtained by the 4-genetransduction were smaller than those by the 10-gene transduction. Whenthese colonies were passaged, the numbers of colonies having iPS cellmorphology was 9 clones among 12 clones in the case of the 10-genetransduction, whereas there was a somewhat lower tendency of 7 clonesamong 12 clones in the case of the 4-gene transduction. As for the 4genes, almost the same numbers of iPS cells were obtained with either ofthose derived from mouse or those derived from human.

When 3 genes selected from the aforementioned 4 genes were transduced,36 flat colonies were obtained with one combination (#14 (Oct3/4), #15(Sox2), and #20 (Klf4)). However, iPS cells were not observed when theywere passaged. With another combination (#14 (Oct3/4), #20 (Klf4), and#22 (c-Myc)), 54 small colonies were obtained. When 6 of the relativelylarge colonies from among those colonies were passaged, cells similar toES cells were obtained for all these 6 clones. However, it seemed thatadhesion of the cells between themselves and to the culture dish wasweaker than that of ES cells. The proliferation rate of the cells wasalso slower than that observed in the case of the transduction with the4 genes. Further, one colony each was formed with each of the other twokinds of combinations of 3 genes among the 4 genes. However,proliferation of the cells was not observed when the cells werepassaged. With any of combinations of 2 genes selected from the 4 genes(6 combinations), no G418-resistant colonies were formed. The aboveresults are shown in FIG. 7.

Further, the results of observation of expression profiles of the EScell marker genes by RT-PCR are shown in FIG. 10. The details of themethod are as follows. From iPS cells established by transducing 3 genes(Oct3/4, Klf4, and c-Myc: represented as “Sox2 minus”), 4 genes (Sox2was added to the three genes: represented as “4ECAT”), and 10 genes (#3,#4, #5, #11, #18, and #21 in TABLE 4 were added to the four genes:represented as “10ECAT”) into Fbx15^(βgeo/βgeo) MEFs; iPS cellsestablished by transducing 10 genes into fibroblasts established fromtail tip of an adult mouse in which βgeo was knocked into the Fbx15 gene(represented as “10ECAT Skin fibroblast”), mouse ES cells, and MEF cellswith no gene transduction, total RNAs were purified, and treated withDNaseI to remove contamination of genomic DNA. First strand cDNAs wereprepared by a reverse transcription reaction, and expression profiles ofthe ES cell marker genes were examined by PCR. For Oct3/4, Nanog, andERas, PCR was performed by using primers which only amplified atranscript product from an endogenous gene, not from the transducedretrovirus. The primer sequences are shown in TABLE 6.

TABLE 6 ECAT1 ECAT1-RT-S TGT GGG GCC CTG AAA GGC GAG CTG AGA T (SEQ IDNO: 1) ECAT1-RT-AS ATG GGC CGC CAT ACG ACG ACG CTC AAC T (SEQ ID NO: 2)Esg1 pH34-U38 GAA GTC TGG TTC CTT GGC AGG ATG (SEQ ID NO: 3) pH34-L394ACT CGA TAC ACT GGC CTA GC (SEQ ID NO: 4) Nanog 6047-S1 CAG GTG TTT GAGGGT AGC TC (SEQ ID NO: 5) 6047-AS1 CGG TTC ATC ATG GTA CAG TC (SEQ IDNO: 6) ERas 45328-S118 ACT GCC CCT CAT CAG ACT GCT ACT (SEQ ID NO: 7)ERas-AS304 CAC TGC CTT GTA CTC GGG TAG CTG (SEQ ID NO: 8) Gdf3 Gdf3-U253GTT CCA ACC TGT GCC TCG CGT CTT (SEQ ID NO: 9) GDF3 L16914 AGC GAG GCATGG AGA GAG CGG AGC AG (SEQ ID NO: 10) Fgf4 Fgf4-RT-S CGT GGT GAG CATCTT CGG AGT GG (SEQ ID NO: 11) Fgf4-RT-AS CCT TCT TGG TCC GCC CGT TCT TA(SEQ ID NO: 12) Cripto Cripto-S ATG GAC GCA ACT GTG AAC ATG ATG TTC GCA(SEQ ID NO: 13) Cripto-AS CTT TGA GGT CCT GGT CCA TCA CGT GAC CAT (SEQID NO: 14) Zfp296 Zfp296-S67 CCA TTA GGG GCC ATC ATC GCT TTC (SEQ ID NO:15) Zfp296-AS350 CAC TGC TCA CTG GAG GGG GCT TGC (SEQ ID NO: 16) Dax1Dax1-S1096 TGC TGC GGT CCA GGC CAT CAA GAG (SEQ ID NO: 17) Dax1-AS1305GGG CAC TGT TCA GTT CAG CGG ATC (SEQ ID NO: 18) Oct3/4 Oct3/4-S9 TCT TTCCAC CAG GCC CCC GGC TC (SEQ ID NO: 19) Oct3/4-AS210 TGC GGG CGG ACA TGGGGA GAT CC (SEQ ID NO: 20) NAT1 NAT1 U283 ATT CTT CGT TGT CAA GCC GCCAAA GTG GAG (SEQ ID NO: 21) NAT1 L476 AGT TGT TTG CTG CGG AGT TGT CATCTC GTC (SEQ ID NO: 22)

The results shown in this figure revealed that, by transduction of the 3genes, expression of each of ERas and Fgf4 was efficiently induced, butexpression of each of Oct3/4 and Nanog, essential factors for themaintenance of pluripotency, was not induced, or was very weak even wheninduced. However, when the 4 genes were transduced, there was one clone(#7) in which Oct3/4 and Nanog were relatively strongly induced among 4clones examined. Further, when the 10 genes were transduced, stronginduction of each of Oct3/4 and Nanog was observed in 3 clones among 5clones examined.

These results revealed that a combination of at least 3 genes (#14(Oct3/4), #20 (Klf4), and #22 (c-Myc)) was essential for reprogrammingunder these conditions, and in the cases of the 4-gene group and 10-genegroup including the 3 kinds of genes, the reprogramming efficiency wasincreased in proportion to the increasing number of genes. In otherwords, in accordance with the guidance disclosed herein, the minimumcombination of nuclear reprogramming factors required for iPS cellinduction under a given set of experimental conditions could be furtheroptimized as evidenced below.

Example 3 Analysis of Pluripotency of Reprogrammed Cells

In order to evaluate the differentiation pluripotency of the establishediPS cells, the iPS cells established with 24 factors, 10 factors, and 4factors were subcutaneously transplanted into nude mice. As a result,tumors having a size similar to that observed with ES cells were formedin all animals. Histologically, the tumors consisted of a plurality ofkinds of cells, and cartilaginous tissues, nervous tissues, musculartissues, fat tissues, and intestinal tract-like tissues were observed(FIG. 8), which verified pluripotency of the iPS cells. In contrast,although tumors were formed when the cells established with the 3factors were transplanted into nude mice, they were formedhistologically only from undifferentiated cells. These results suggestedthat a Sox family gene was essential for the induction ofdifferentiation pluripotency.

Example 4 Reprogramming of Fibroblasts Derived from Tails of Adult Mice

The 4 factors identified in the mouse embryonic fibroblasts (MEFs) weretransduced into fibroblasts derived from tails of βgeo knockin Fbx15adult mice systemically expressing green fluorescence protein (GFP).Then, the cells were cultured on feeder cells under the same conditionsas ES cell culture conditions, and G418 selection was performed. Inabout two weeks after the start of the drug selection, a plurality ofcolonies of iPS cells were obtained. When these cells weresubcutaneously transplanted to nude mice, teratomas consisting of avariety of all three germ layer tissues were formed. Further, when theiPS cells derived from adult dermal fibroblasts were transplanted to theblastocysts, and then transplanted into the uteri of pseudopregnantmice, embryos in which the GFP-positive cells were systemicallydistributed were observed among those at 13.5 days after fertilization(FIG. 9), demonstrating that the iPS cells had pluripotency and wereable to contribute to mouse embryogenesis. These results indicate thatthe identified class of factors had an ability to induce reprogrammingof not only somatic cells in an embryonic period, but also somatic cellsof mature mice. Practically, it is extremely important that thereprogramming can be induced in cells derived from adult skin.

Example 5 Effect of Cytokine on iPS Cell Establishment

An effect of cytokine on iPS cell establishment was investigated.Expression vector (pMX retroviral vector) for basic fibroblast growthfactor (bFGF) or stem cell factor (SCF) was transduced into feeder cells(STO cells) to establish cells permanently expressing the cytokines.MEFs derived from the Fbx15^(βgeo/βgeo) mouse (500,000 cells/100 mmdish) were cultured on these STO cells and transduced with the 4factors, and then subjected to G418 selection. As a result, the numberof formed colonies increased 20 times or higher on the STO cellsproducing bFGF (FIG. 11) or SCF (data not shown), as compared with theculture on normal STO cells. Further, although no iPS cell colony wasformed on the normal STO cells when the 3 factors other than c-Myc weretransduced, colony formation was observed on the STO cells producingbFGF (FIG. 11) or SCF (data not shown). These results revealed thatstimulation with the cytokine increased the efficiency of theestablishment of iPS cells from MEFs, and the nuclear reprogramming wasachievable by using a cytokine instead of c-Myc.

Example 6 iPS Cell Generation with Other Oct, Klf, Myc, and Sox FamilyMembers

Family genes exist for all of the Oct3/4, Klf4, c-Myc, and Sox2 genes(TABLES 1 and 2). Accordingly, studies were made as to whether iPS cellscould be established with the family genes instead of the 4 genes. InTABLE 7, combined experimental results in duplicate are shown. Withregard to the Sox family, Sox1 gave almost the same number ofG418-resistant colonies formed and iPS cell establishment efficiency asthose with Sox2. As for Sox3, the number of G418-resistant coloniesformed was about 1/10 of that with Sox2, however, iPS cell establishmentefficiency of the colonies picked up was in fact higher than that withSox2. As for Sox 15, both the number of G418-resistant colonies formedand iPS cell establishment efficiency were lower than those with Sox2.As for Sox 17, the number of G418-resistant colonies formed was almostthe same as that with Sox2, however, iPS cell establishment efficiencywas low. With regard to the Klf family, Klf2 gave a smaller number ofG418-resistant colonies than Klf4, however, they gave almost the sameiPS cell establishment efficiency. With regard to the Myc family, it wasfound that wild-type c-Myc was almost the same as a T58A mutant both inthe number of G418-resistant colonies formed and iPS cell establishmentefficiency. Further, each of N-Myc and L-Myc (each wild type) was almostthe same as c-Myc in both of the number of G418-resistant coloniesformed and iPS cell establishment efficiency.

TABLE 7 Number of Number of Number of iPS cell Transduced formed pickedestablished iPS establishment gene colonies colonies cell strainefficiency (%) 4 Factors 85 12 5 42 (cMycT58A) Sox1 84 12 7 58 Sox3 8 87 92 Sox15 11 11 1 8 Sox17 78 12 2 17 Klf2 11 10 5 50 c-MycWT 53 11 8 72N-MycWT 40 12 7 58 L-MycWT 50 12 11 92 3 Factors 6 6 2 17 (-Sox2)

Example 7 Use of a Nanog-GFP-Puro^(r) Reporter to Establish iPS Cells

Studies were made as to whether iPS cells could be established with areporter other than Fbx15-βgeo. Escherichia coli artificial chromosome(BAC) containing the Nanog gene in the center was isolated, and then theGFP gene and the puromycin resistance gene were knocked in byrecombination in E. coli (FIG. 12A). Subsequently, the above modifiedBAC was introduced into ES cells to confirm that the cells becameGFP-positive in an undifferentiated state specific manner (data notshown). Then, these ES cells were transplanted in mouse blastocysts tocreate transgenic mice via chimeric mice. In these mice, GFP-positivecells were specifically observed in inner cell masses of the blastocystsor gonads of embryos at 13.5 days after fertilization (FIG. 12B). Thegonads were removed from the embryos at 13.5 days after fertilization(hybrid of DBA, 129, and C57BL/6 mice), and MEFs were isolated. Theisolated MEFs were confirmed to be GFP-negative (FIG. 13) by flowcytometry. These MEFs were retrovirally transduced with the 4 factorsand subjected to puromycin selection, and as a result, a plural numberof resistant colonies were obtained. Only about 10 to 20% of thecolonies were GFP-positive. When the GFP-positive colonies werepassaged, they gave morphology (FIG. 14) and proliferation (FIG. 15)similar to those of ES cells. Examination of the gene expression patternrevealed that the expression pattern was closer to that of ES cells ascompared to the iPS cells isolated from Fbx15^(geo/βgeo) MEFs by G418selection (FIG. 16). When these cells were transplanted to nude mice,teratoma formation was induced, thereby the cells were confirmed to beiPS cells (FIG. 17). Further, chimeric mice were born by transplantingthe iPS cells obtained by Nanog-GFP selection to the blastocysts ofC57BL/6 mice (FIG. 18). When these chimeric mice were mated, germ-linetransmission was observed (FIG. 19). In these iPS cells established byNanog-GFP selection, which were closer to ES cells, the expressions ofthe 4 factors from the retroviruses were almost completely silenced,suggesting that self-replication was maintained by endogenous Oct3/4 andSox2.

Example 8 In Vitro Differentiation Induction

Confluent iPS cells in 10 cm dishes were trypsinized and suspended in EScell medium (the STO cells were removed by adhesion to a gelatin-coateddish for 10 to 20 minutes after the suspension). 2×10⁶ cells werecultured for four days in a HEMA (2-hydroxyethyl methacrylate) coated E.coli culture dish as a suspension culture to form embryoid bodies (EBs)(day 1 to 4). On the 4th day of EB formation (day 4), all of the EBswere transferred to a 10-cm tissue culture dish, and cultured in ES cellmedium for 24 hours to allow adhesion. After 24 hours (day 5), themedium was changed to an ITS/fibronectin-containing medium. The culturewas performed for 7 days (medium was exchanged every 2 days), andnestin-positive cells were selected (cells of other pedigrees were dyingto some extent in a culture under serum-free condition)(day 5 to 12).A2B5-positive cells were then induced. After 7 days (day 12), the cellswere separated by trypsinization, and the remaining EBs were removed.1×10⁵ cells were seeded on a poly-L-ornithine/fibronectin-coated 24-wellplate, and cultured for 4 days in an N2/bFGF-containing medium (mediumwas exchanged every 2 days)(day 12 to 16). After 4 days (day 16), themedium was changed to an N2/bFGF/EGF-containing medium, and the culturewas continued for 4 days (medium was exchanged every 2 days)(day 16 to20). After 4 days (day 20), the medium was changed to anN2/bFGF/PDGF-containing medium, and the culture was continued for 4 days(medium was exchanged every 2 days)(day 20 to 24). During this period(day 12 to 24), when the cells had increased excessively and reachedconfluent, they were passaged at appropriate times, and 1 to 2×10⁵ cellswere seeded (the number of the cells varied depending on the timing ofthe passage). After 4 days (day 24), the medium was changed to an N2/T3medium, and the culture was continued for 7 days (day 24 to 31) withmedium exchange every 2 days. On day 31, the cells were fixed andsubjected to immunostaining. As a result, differentiation of the iPScells into βIII tubulin-positive nerve cells, O4-positiveoligodendrocytes, and GFAP-positive astrocytes was observed (FIG. 20).

Example 9 Establishment of iPS Cells Without Drug Selection

In order to establish iPS cells from arbitrary mouse somatic cells otherthan those derived from the Fbx15-βgeo knockin mouse, a method for theestablishment without using drug selection was developed. 10,000,50,000, or 100,000 cells mouse embryo fibroblasts (MEFs) were culturedon a 10 cm dish (on STO feeder cells). This is less than the number ofcells used above, Control DNA or the 4 factors were retrovirallytransduced. When culture was performed for 2 weeks in the ES cell medium(without G418 selection), no colony formation was observed in the dishin which the control DNA was transduced, whilst in the dish in which the4 factors were transduced, a plurality of compact colonies were formedas well as flat colonies considered to be transformed (FIG. 21). When 24colonies were picked up from these colonies and culture was continued,ES cell-like morphology was observed. Gene expression profiles thereofwere examined by RT-PCR, and as a result, the expression of Esg1, an EScell marker, was observed in 7 clones. Induction of many ES cell markerssuch as Nanog, ERas, GDF3, Oct3/4, and Sox2 was observed in clone 4, andtherefore the cells were considered to be iPS cells (FIG. 22). The aboveresults demonstrated that drug selection using Fbx15-βgeo knockin or thelike was not indispensable for iPS cell establishment, and iPS cellscould be established from arbitrary mouse-derived somatic cells. Thisalso suggested the possibility that iPS cells could be established fromsomatic cells of a disease model mouse by the aforementioned technique.

Example 10 iPS Cell Generation from Hepatocytes and Gastric Mucous Cells

As cells from which iPS cells were induced, hepatocytes and gastricmucous cells being cells other than fibroblasts were examined.Hepatocytes were isolated from the liver of the Fbx15^(βgeo/βgeo) miceby perfusion. These hepatocytes were retrovirally introduced with the 4factors, and then subjected to G418 selection to obtain plural iPS cellcolonies. As a result of gene expression pattern analysis using a DNAmicroarray, the iPS cells derived from the liver were found to be moresimilar to ES cells than the iPS cells derived from dermal fibroblastsor embryonic fibroblasts. iPS cells were obtained also from gastricmucous cells in the same manner as those from hepatocytes.

Example 11 Effect of MAP Kinase Inhibitor on iPS Cell Establishment

PD98059 is an inhibitor of MAP kinase which suppresses proliferation ofvarious differentiated cells. However, it is known to promotemaintenance of undifferentiated status and proliferation of ES cells.Effects of PD98059 on iPS cell establishment were thus examined. MEFsestablished from a mouse having the selective markers ofNanog-EGFP-IRES-Puro were retrovirally introduced with the 4 factors andsubjected to puromycin selection. When PD98059 was not added, thepercentage of GFP-positive colonies was 8% of the iPS cell coloniesobtained. However, in the group to which PD98059 (final concentration:25 μM) was continuously added from the next day of the retroviraltransfection, 45% of the colonies obtained were GFP-positive. Theresults were interpreted to be due to PD98059 promoting theproliferation of the GFP-positive iPS cells, which are closer to EScells, whilst PD98059 suppressing the proliferation of the GFP-negativeiPS cells or differentiated cells. From these results, PD98059 wasdemonstrated to be able to be used for establishment of the iPS cellscloser to ES cells or establishment of iPS cells without using drugselection.

Example 12 Establishment of iPS Cells from Embryonic HDFs in Mouse ESCell Medium

A plasmid, containing the red fluorescence protein gene downstream fromthe mouse Oct3/4 gene promoter and the hygromycin resistance genedownstream from the PGK promoter, was introduced by nucleofection intoembryonic human dermal fibroblasts (HDFs) in which solute carrier family7 (Slc7a1, NCBI accession number NM_(—)007513) as a mouse ecotropicvirus receptor was expressed by lentiviral transduction. Hygromycinselection was performed to establish strains with stable expression.800,000 cells were seeded on the STO cells treated with mitomycin, andon the next day, Oct3/4, Sox2, Klf4, and c-Myc (each derived from human)were retrovirally transduced into the cells. 24 colonies were picked upfrom those obtained after 3 weeks (FIG. 23, left), and transferred on a24-well plate on which the STO cells were seeded and then cultured.After 2 weeks, one grown clone was passaged on a 6-well plate on whichthe STO cells were seeded and cultured. As a result, cellsmorphologically similar to ES cells were obtained (FIG. 23, right),suggesting that the cells were iPS cells. The mouse ES cell medium wasused as every medium.

Example 13 Establishment of iPS Cells from Adult HDFs in Mouse ES CellMedium

Human adult dermal fibroblasts (adult HDFs) or human neonatal foreskincells (BJ) were transduced with Slc7a1 (mouse retroviral receptor) byusing lentivirus, and the resulting cells were seeded on 800,000 feedercells (mitomycin-treated STO cells). The genes were retrovirallytransduced as the following combinations.

1. Oct3/4, Sox2, Klf4, c-Myc, TERT, and SV40 Large T antigen

2. Oct3/4, Sox2, Klf4, c-Myc, TERT, HPV16 E6

3. Oct3/4, Sox2, Klf4, c-Myc, TERT, HPV16 E7

4. Oct3/4, Sox2, Klf4, c-Myc, TERT, HPV16 E6, HPV16 E7

5. Oct3/4, Sox2, Klf4, c-Myc, TERT, Bmi1

(Oct3/4, Sox2, Klf4, c-Myc and TERT were derived from human, and Bmi1was derived from mouse)

The culture was continued under the culture conditions for mouse EScells without drug selection. As a result, colonies considered to bethose of iPS cells emerged on the 8th day after the virus transfectionon the dish in which the factors were introduced according toCombination 1 (FIG. 24). iPS cell-like colonies also emerged with theother combinations (2 to 5), although they were not as apparent whencompared to Combination 1. When only the 4 factors were transduced, nocolonies emerged. Cells transduced with only the four factors under theexperimental conditions used in this Example showed only faint stainingfor alkaline phosphatase (FIG. 25(A)-(B)).

However, optimization of the methods heretofore described revealedsuccessful induction of iPS cells through staining of any number ofpluripotent markers, including alkaline phosphatase, ABCG-2, E-cadherin,SSEA-3, and SSEA-4 when adult human dermal fibroblasts expressing mouseSlc7a1 gene were generated into iPS cells by reprogramming with the fourfactors plus TERT and SV40 Large T antigen (i.e. six factors total:c-Myc, Klf4, Sox2, Oct3/4, TERT, and SV40 Large T antigen) (FIG.26(A)-(B)). These cells were assessed for pluripotent cell markers (FIG.27). These cells were found to express ECATS, including Nanog and ESG1.Similarly, BJ fibroblasts expressing mouse Slc7a1 gene were generatedinto iPS cells by reprogramming with the following same factors. Thesecells were also tested for pluripotent cell markers (FIG. 28). Inaddition, iPS cells generated from adult HDFs were selected forsubcutaneous injection into the dorsal flanks of SCID mice. Teratomaformation was observed (FIGS. 29(A)-(D)). Human dermal fibroblasts werealso shown to differentiate in vitro upon culturing in HEMA-coatedplates (7 days) and gelatinized dishes (7 days) (FIG. 30).

Example 14 Optimization of Retroviral Transduction for Generating HumaniPS Cells

Next, iPS cell generation from adult human somatic cells was furtherevaluated by optimizing retroviral transduction in human fibroblasts andsubsequent culture conditions. Induction of iPS cells from mousefibroblasts requires retroviruses with high transduction efficiencies(Takahashi et al., Cell 126: 663-676, 2006). Therefore, transductionmethods in adult human dermal fibroblasts (HDFs) were optimized. First,green fluorescent protein (GFP) was introduced into adult HDF withamphotropic retrovirus produced in PLAT-A packaging cells. As a control,GFP was introduced into mouse embryonic fibroblasts (MEF) with ecotropicretrovirus produced in PLAT-E packaging cells (Morita et al., Gene Ther.7:1063-66, 2000). In MEF, more than 80% of cells expressed GFP (FIG.31). In contrast, less than 20% of HDF expressed GFP with significantlylower intensity than in MEF. To improve the transduction efficiency, themouse receptor for retroviruses, Slc7a1 (Verrey et al., Pflugers Arch.447:532-542, 2004) (also known as mCAT1), was introduced into HDF withlentivirus. Then GFP was introduced into HDF-Slc7a1 with ecotropicretrovirus. This strategy yielded a transduction efficiency of 60%, witha similar intensity to that in MEF.

Example 15 Generation of iPS Cells from Adult HDFs in Primate ES CellCulture Medium

The protocol for human iPS cell induction is summarized in FIG. 32A.pMXs encoding human Oct3/4, Sox2, Klf4, and c-Myc were introduced intoHDF-Slc7a1 cells (FIG. 32B; 8×10⁵ cells per 100 mm dish). The HDFs werederived from facial dermis of 36-year-old Caucasian female.

Six days after transduction, the cells were harvested by trypsinizationand plated onto mitomycin C-treated SNL feeder cells (McMahon et al.,Cell 62:1073-85, 1990) at 5×10⁴ or 5×10⁵ cells per 100 mm dish. The nextday, the medium (DMEM containing 10% FBS) was replaced with a medium forprimate ES cell culture supplemented with 4 ng/ml basic fibroblastgrowth factor (bFGF). Approximately two weeks later, some granulatedcolonies appeared that were not similar to hES cells in morphology (FIG.32C). Around day 25, distinct types of colonies that were flat andresembled hES cell colonies were observed (FIG. 32D). From 5×10⁴fibroblasts, ˜10 hES cell-like colonies and ˜100 granulated colonies(7/122, 8/84, 8/171, 5/73, 6/122, and 11/213 in six independentexperiments, summarized in TABLE 8) were observed.

TABLE 8 Summary of the iPS cell induction experiments Cell No. No. ofNo. of No. of No. of Exp. Parental seeded at ES-like total picked upestablished ID cells d6 colony colony colony clone 201B HDF 50000 7 1297 5 243H HFLS 500000 0 >1000 50000 17 679 6 2 246B HDF 500000 0 420500000 2 508 50000 8 92 6 6 246G BJ 50000 7 10 6 5 500000 86 98 500000106 108 249D HDF 500000 0 320 500000 0 467 50000 8 179 6 4 253F HDF50000 5 78 3 2 50000 6 128 3 3 500000 10 531 500000 3 738 282C HDF 5000011 224 3 1 282H BJ 50000 13 15 3 2 282R HFLS 5000 31 98 6 2

At day 30, hES cell-like colonies were picked up and mechanicallydisaggregated into small clumps without enzymatic digestion. Whenstarting with 5×10⁵ fibroblasts, the dish was nearly covered with morethan 300 granulated colonies. Occasionally some hES cell-like coloniesin between the granulated cells were observed, but it was difficult toisolate hES cell-like colonies because of the high density of granulatedcells.

The hES-like cells expanded on SNL feeder cells under the human ES cellculture condition. They formed tightly packed and flat colonies (FIG.32E). Each cell exhibited morphology similar to that of human ES cells,characterized by large nucleoli and scant cytoplasm (FIG. 32F). As isthe case with hES cells, occasionally spontaneous differentiation wasobserved in the center of the colony (FIG. 32G).

These cells also showed similarity to hES cells in feeder dependency.They did not attach to gelatin-coated tissue-culture plates. Bycontrast, they maintained an undifferentiated state on Matrigel-coatedplates in MEF-conditioned medium (MEF-CM), but not in ES medium (FIG.33).

Since these cells were indistinguishable from hES cells in morphologyand other aspects noted above, the selected cells after transduction ofHDFs are referred to as human iPS cells. The molecular and functionalevidence for this claim is further described below. Human iPS cellsclones established in this study are summarized in TABLE 9.

TABLE 9 Characterization of established clones Marker Pluripotencyexpression Cardio- Clone Source RT-PCR IC EB PA6 myocyte Teratoma 201B1HDF ✓ 201B2 ✓ ✓ ✓ ✓ ✓ 201B3 ✓ 201B6 ✓ ✓ ✓ ✓ ✓ 201B7 ✓ ✓ ✓ ✓ ✓ ✓ 243H1HFLS ✓ ✓ 243H7 ✓ ✓ 246B1 HDF ✓ 246B2 ✓ 246B3 ✓ 246B4 ✓ 246B5 ✓ 246B6 ✓246G1 BJ ✓ ✓ 246G3 ✓ ✓ 246G4 ✓ 246G5 ✓ 246G6 ✓ 253F1 HDF ✓ 253F2 ✓ 253F3✓ 253F4 ✓ 253F5 ✓ IC; immunocytochemistry, EB; embryoid body

Human iPS Cells Express hES Markers

In general, except for a few cells at the edge of the colonies, humaniPS cells did not express stage-specific embryonic antigen (SSEA)-1(FIG. 32H). In contrast, they expressed hES cell-specific surfaceantigens (Adewumi et al., Nat. Biotechnol. 25:803-816, 2007), includingSSEA-3, SSEA-4, tumor-related antigen (TRA)-1-60, TRA-1-81, andTRA-2-49/6E (alkaline phosphatase), and NANOG protein (FIG. 32I-N).

RT-PCR showed human iPS cells expressed many undifferentiated ES cellgene markers (Adewumi et al., Nat. Biotechnol. 25:803-816, 2007), suchas OCT3/4, SOX2, NANOG, growth and differentiation factor 3 (GDF3),reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4),embryonic cell-specific gene 1 (ESG1), developmentalpluripotency-associated 2 (DPPA2), DPPA4, and telomerase reversetranscriptase (hTER7) at levels equivalent to or higher than those inthe human embryonic carcinoma cell line, NTERA-2 (FIG. 34A). By westernblotting, proteins levels of OCT3/4, SOX2, NANOG, SALL4, E-CADHERIN, andhTERT were similar in human iPS cells and hES cells (FIG. 34B). In humaniPS cells, the expression of transgenes from integrated retroviruses wasefficiently silenced, indicating that they depend on the endogenousexpression of these genes (FIG. 34C).

Promoters of ES Cell-Specific Genes are Active in Human iPS Cells

Bisulfite genomic sequencing analyses evaluating the methylationstatuses of cytosine guanine dinucleotides (CpG) in the promoter regionsof pluripotent-associated genes, such as OCT3/4, REX1, and NANOG,revealed that they were highly unmethylated, whereas the CpGdinucleotides of the regions were highly methylated in parental HDFs(FIG. 34D). These findings indicate that these promoters are active inhuman iPS cells.

Luciferase reporter assays also showed that human OCT3/4 and REX1promoters had high levels of transcriptional activity in human iPScells, but not in HDF. The promoter activities of ubiquitously expressedgenes, such as human RNA polymerase II (PolII), showed similaractivities in both human iPS cells and HDF (FIG. 34E).

High Telomerase Activity and Exponential Growth of Human iPS Cells

As predicted from the high expression levels of hTERT, human iPS cellsshowed high telomerase activity (FIG. 35A). They proliferatedexponentially for at least 4 months (FIG. 35B). The calculated doublingtime of human iPS cells were 46.9±12.4 (clone 201B2), 47.8±6.6 (201B6)and 43.2±11.5 (201B7) hours (FIG. 35B). These times are equivalent tothe reported doubling time of hES cells (Cowan et al., N. Engl. J. Med.350:1353-56, 2004).

Human iPS Cells Are Derived from HDF, not Cross-Contamination

PCR of genomic DNA of human iPS cells showed that all clones haveintegration of all the four retroviruses (FIG. 36A). Southern blotanalysis with a c-Myc cDNA probe revealed that each clone had a uniquepattern of retroviral integration sites (FIG. 36B). In addition, thepatterns of 16 short tandem repeats were completely matched betweenhuman iPS clones and parental HDF (TABLE 10).

TABLE 10 STR analyses of HDF-derived iPS cells Clone Locus 201B1 201B2201B3 201B6 201B7 NTERA-2 HDF D3S1358 15 17 15 17 15 17 15 17 15 17 1515 17 TH01 5 5 5 5 5 9 5 D21S11 28 28 28 28 28 29 30 28 D18S51 14 14 1414 14 13 14 Penta_E 7 19 7 19 7 19 7 19 7 19 5 14 7 19 D5S818 11 11 1111 11 8 11 11 D13S317 10 14 10 14 10 14 10 14 10 14 14 10 14 D7S820 9 109 10 9 10 9 10 9 10 12 9 10 D16S539 11 13 11 13 11 13 11 13 11 13 11 1611 13 CSF1PO 10 10 10 10 10 9 11 10 Penta_D 8 10 8 10 8 10 8 10 8 10 1112 8 10 AMEL X X X X X X Y X vWA 15 18 15 18 15 18 15 18 15 18 19 15 18D8S1179 8 10 8 10 8 10 8 10 8 10 13 15 8 10 TPOX 8 9 8 9 8 9 8 9 8 9 8 98 9 FGA 20 22 20 22 20 22 20 22 20 22 23 20 22 These patterns differedfrom any established hES cell lines reported on National Institutes ofHealth website(http://stemcells.nih.gov/research/nihresearch/scunit/genotyping.htm).In addition, chromosomal G-band analyses showed that human iPS cells hada normal karyotype of 46XX (not shown). Thus, human iPS clones werederived from HDF and were not a result of cross-contamination.

Example 16 Embryoid Body-Mediated Differentiation of Human iPS Cells

To determine the differentiation ability of human iPS cells in vitro,floating cultivation was used to form embryoid bodies (EBs)(Itskovitz-Eldor et al., Mol. Med. 6:88-95, 2000). After 8 days insuspension culture, iPS cells formed ball-shaped structures (FIG. 37A).These embryoid body-like structures were transferred to gelatin-coatedplates and continued cultivation for another 8 days. Attached cellsshowed various types of morphologies, such as those resembling neuronalcells, cobblestone-like cells, and epithelial cells (FIG. 37B-E).Immunocytochemistry detected cells positive for βIII-tubulin (a markerof ectoderm), glial fibrillary acidic protein (GFAP, ectoderm), α-smoothmuscle actin (α-SMA, mesoderm), desmin (mesoderm), α-fetoprotein (AFP,endoderm), and vimentin (mesoderm and parietal endoderm) (FIG. 37F-K).RT-PCR confirmed that these differentiated cells expressed forkhead boxA2 (FOXA2, a marker of endoderm), AFP (endoderm), cytokeratin 8 and 18(endoderm), SRY-box containing gene 17 (SOX17, endoderm), BRA CHYURY(mesoderm), Msh homeobox 1 (MSX1, mesoderm), microtubule-associatedprotein 2 (MAP2, ectoderm), and paired box 6 (PAX6, ectoderm) (FIG.37L). In contrast, expression of OCT3/4, SOX2, and NANOG wassignificantly decreased. These data demonstrated that iPS cells coulddifferentiate into three germ layers in vitro.

Example 17 Directed Differentiation of Human iPS Cells into Neural Cells

Next, it was examined whether lineage-directed differentiation of humaniPS cells could be induced by reported methods for hES cells. Human iPScells were seeded on PA6 feeder layer and maintained underdifferentiation conditions for 2 weeks (Kawasaki et al., Nueron28:31-40, 2000). Cells spread drastically, and some neuronal structureswere observed (FIG. 38A). Immunocytochemistry detected cells positivefor tyrosine hydroxylase and βIII tubulin in the culture (FIG. 38B). PCRanalysis revealed expression of dopaminergic neuron markers, such asaromatic-L-amino acid decarboxylase (AADC), choline acetyltransferase(ChAT), solute carrier family 6 (neurotransmitter transporter,dopamine), member 3 (DAT), and LIM homeobox transcription factor 1 beta(LMX1B), as well as another neuron marker, MAP2 (FIG. 38C). In contrast,GFAP expression was not induced with this system. On the other hand,expression of OCT3/4, SOX2, and NANOG decreased (FIG. 38C). These datademonstrated that iPS cells could differentiate into neuronal cells,including dopaminergic neurons, by co-culture with PA6 cells.

Example 18 Directed Differentiation of Human iPS Cells into CardiacCells

Next directed cardiac differentiation of human iPS cells was examinedwith the recently reported protocol, which utilizes activin A and bonemorphogenetic protein (BMP) 4 (Laflamme et al., Nat. Biotechnol.25:1015-24, 2007). Twelve days after the induction of differentiation,clumps of cells started beating (FIG. 38D). RT-PCR showed that thesecells expressed cardiomyocyte markers, such as troponin T type 2 cardiac(TnTc); myocyte enhancer factor 2C (MEF2C); NK2 transcription factorrelated, locus 5 (NKX2.5) myosin, light polypeptide 7, regulatory(MYL2A), and myosin, heavy polypeptide 7, cardiac muscle, beta (MYHCB)(FIG. 38E). In contrast, the expression of Oct3/4, Sox2, and Nanogmarkedly decreased. Thus, human iPS cells can differentiate into cardiacmyocytes in vitro.

Example 19 Teratoma Formation from Human iPS Cells

To test pluripotency in vivo, human iPS cells (clone 201B7) weretransplanted subcutaneously into dorsal flanks of immunodeficient (SCID)mice. Nine weeks after injection, tumor formation was observed.Histological examination showed that the tumor contained various tissues(FIG. 39), including gut-like epithelial tissues (endoderm), striatedmuscle (mesoderm), cartilage (muscle), neural tissues (ectoderm), andkeratin-containing squamous tissues (ectoderm).

Example 20 Generation of iPS Cells from Other Human Somatic Cells

In addition to HDF, primary human fibroblast-like synoviocytes (HFLS)from synovial tissue of 69-year-old Caucasian male and BJ cells, a cellline established from neonate fibroblasts, were used (TABLE 8). From5×10⁴ HFLS cells per 100 mm dish, more than 600 hundred granulatedcolonies and 17 hES cell-like colonies were obtained. Six colonies werepicked, of which only two were expandable as iPS cells (FIG. 40). Dishesseeded with 5×10⁵ HFLS were covered with granulated cells, and no hEScell-like colonies were distinguishable. In contrast, 7 to 8 and ˜100hES cell-like colonies were obtained from 5×10⁴ and 5×10⁵ BJ cells,respectively, with only a few granulated colonies (TABLE 8). Six hEScell-like colonies were picked and iPS cells were generated from fivecolonies (FIG. 40). Human iPS cells derived from HFLS and BJ expressedhES cell-marker genes at levels similar to or higher than those in hEScells (FIG. 41). They differentiated into all three germ layers throughEBs (FIG. 42). STR analyses confirmed that iPS-HFLS cells and iPS-BJcells were derived from HFLS and BJ fibroblasts, respectively (TABLE 11and TABLE 12).

TABLE 11 STR analyses of HFLS-derived IPS cells Clone Locus 243H1 243H7HFLS D3S1358 16 17 16 17 16 17 TH01 5 9 5 9 5 9 D21S11 28 30 28 30 28 30D18S51 14 17 14 17 14 17 Penta_E 5 12 5 12 5 12 D5S818 10 12 10 12 10 12D13S317 13 13 13 D7S820 9 12 9 12 8 12 D16S539 11 13 11 13 11 13 CSF1PO10 11 10 11 10 11 Penta_D 9 11 9 11 9 11 AMEL X X Y X Y vWA 17 19 17 1917 19 D8S1179 13 13 13 TPOX 8 11 8 11 8 11 FGA 21 22 21 22 21 22

TABLE 12 STR analyses of BJ-derived iPS cells Clone Locus 246G1 246G3246G4 246G5 246G6 BJ D3S1358  13 15 13 15  13 15 13 15 13 15 13 15 TH016 7 6 7 6 7 6 7 6 7 6 7 D21S11 28 28 28 28 28 28 D18S51 16 18 16 18 1618 16 18 16 18 16 18 Penta_E 7 17 7 17 7 17 7 17 7 17 7 17 D5S818 11 1111 11 11 11 D13S317 9 10 9 10 9 10 9 10 9 10 9 10 D7S820 11 12 11 12 1112 11 12 11 12 11 12 D16S539 9 13 9 13 9 13 9 13 9 13 9 13 CSF1PO 9 11 911 9 11 9 11 9 11 9 11 Penta_D 11 12 11 12 11 12 11 12 11 12 11 12 AMELX Y X Y X Y X Y X Y X Y vWA 16 18 16 18 16 18 16 18 16 18 16 18 D8S11799 11 9 11 9 11 9 11 9 11 9 11 TPOX 10 11 10 11 10 11 10 11 10 11 10 11FGA 22 23 22 23 22 23 22 23 22 23 22 23

Thus, with Examples 13-20 it was shown that iPS cells can be generatedfrom adult HDF and other somatic cells by retroviral transduction of thesame four transcription factors, namely Oct3/4, Sox2, Klf4, and c-Myc.The established human iPS cells are indistinguishable from hES cells inmany aspects, including morphology, proliferation, feeder dependence,surface markers, gene expression, promoter activities, telomeraseactivities, in vitro differentiation, and teratoma formation. The fourretroviruses are nearly completely silenced in human iPS cells,indicating that these cells are fully reprogrammed and do not depend oncontinuous expression of the transgenes for self-renewal.

hES cells are different from mouse counterparts in many respects (Rao,M., Dev. Biol. 275:269-286, 2004). hES cell colonies are flatter and donot override each other. hES cells depend on bFGF for self renewal (Amitet al., Dev. Biol. 227:271-78, 2000), whereas mouse ES cells depend onthe LIF/Stat3 pathway (Matsuda et al., EMBO J. 18:4261-69, 1999; Niwa etal., Genes Dev. 12:2048-60, 1998). BMP induces differentiation in hEScells (Xu et al., Nat. Methods 2:185-90, 2005) but is involved in selfrenewal of mouse ES cells (Ying et al., Cell 115:281-92, 2003). Becauseof these differences, it has been speculated that factors required forreprogramming might differ between humans and mice. On the contrary, ourdata show that the same four transcription factors induce iPS cells inboth humans and mouse. The four factors, however, could not induce humaniPS cell colonies when fibroblasts were kept under the culture conditionfor mouse ES cells after retroviral transduction (See Example 13,above), even though these cells stained positive for alkalinephosphatase. These data suggest that the fundamental transcriptionalnetwork governing pluripotency is common in human and mice, butextrinsic factors and signals maintaining pluripotency are unique foreach species.

Deciphering of the mechanism by which the four factors inducepluripotency in somatic cells remains elusive. The function of Oct3/4and Sox2 as core transcription factors to determine pluripotency is welldocumented (Boyer et al., Cell 122:947-956, 2005; Loh et al., Nat Genet.38:431-440, 2006; Wang et al., Nature 444:364-368, 2006). Theysynergistically upregulate “stemness” genes, while suppressingdifferentiation-associated genes in both mouse and human ES cells.However, they cannot bind their targets genes in differentiated cells,because of other inhibitory mechanisms, including DNA methylation. Itmay be speculated that c-Myc and Klf4 modifies chromatin structure sothat Oct3/4 and Sox2 can bind to their targets (Yamanaka, Cell Stem Cell1:39-49, 2007). Notably, Klf4 interacts with p300 histoneacetyltransferase and regulates gene transcription by modulating histoneacetylation (Evans et al., J Biol Chem, 2007).

The negative role of c-Myc in the self renewal of hES cells has alsobeen reported (Sumi et al., Oncogene 26: 5564-5576, 2007). They showedthat forced expression of c-Myc induced differentiation and apoptosis ofhuman ES cells. During iPS cell generation, transgenes derived fromretroviruses are silenced when the transduced fibroblasts acquireES-like state. The role of c-Myc in establishing iPS cells may be as abooster of reprogramming, rather than a controller of maintenance ofpluripotency.

It has been found that each iPS clone contained 3-6 retroviralintegrations for each factor. Thus, each clone had more than 20retroviral integration sites in total, which may increase the risk oftumorigenesis. In the case of mouse iPS cells, ˜20% of chimera mice andtheir offspring derived from iPS cells developed tumors Okita et al.,Nature 448:313-17, 2007). This issue must be overcome to use iPS cellsin human therapies. Therefore, non-retroviral methods to introduce thefour factors, such as adenoviruses or cell-permeable recombinantproteins, are also contemplated as part of the invention. Alternatively,small molecules may replace the four factors for the induction of iPScells.

Experimental Procedures for Examples 14-20 Cell Culture

HDFs from facial dermis of 36-year-old Caucasian female and HFLS fromsynovial tissue of 69-year-old Caucasian male were purchased from CellApplications, Inc. BJ fibroblasts from neonatal foreskin and NTERA-2clone D1 human embryonic carcinoma cells were obtained from AmericanType Culture Collection. Human fibroblasts, NTERA-2, PLAT-E, and PLAT-Acells were maintained in Dulbecco's modified eagle medium (DMEM, NacalaiTesque, Japan) containing 10% fetal bovine serum (FBS, Japan Serum) and0.5% penicillin and streptomycin (Invitrogen). 293FT cells weremaintained in DMEM containing 10% FBS, 2 mM L-glutamine (Invitrogen),1×10⁻⁴ M nonessential amino acids (Invitrogen), 1 mM sodium pyruvate(Sigma) and 0.5% penicillin and streptomycin. PA6 stroma cells (RIKENBioresource Center, Japan) were maintained in α-MEM containing 10% FBSand 0.5% penicillin and streptomycin. iPS cells were generated andmaintained in Primate ES medium (ReproCELL, Japan) supplemented with 4ng/ml recombinant human basic fibroblast growth factor (bFGF, WAKO,Japan). For passaging, human iPS cells were washed once with PBS andthen incubated with DMEM/F12 containing 1 mg/ml collagenase IV(Invitrogen) at 37° C. When colonies at the edge of the dish starteddissociating from the bottom, DMEF/F12/collangenase was removed andwashed with hES cell medium. Cells were scraped and collected into 15 mlconical tube. An appropriate volume of the medium was added, and thecontents were transferred to a new dish on SNL feeder cells. The splitratio was routinely 1:3. For feeder-free culture of iPS cells, the platewas coated with 0.3 mg/ml Matrigel (growth-factor reduced, BDBiosciences) at 4° C. overnight. The plate was warmed to roomtemperature before use. Unbound Matrigel was aspirated off and washedout with DMEM/F12. iPS cells were seeded on Matrigel-coated plate inMEF-CM or ES medium, both supplemented with 4 ng/ml bFGF. The medium waschanged daily. For preparation of MEF-CM, MEFs derived from embryonicday 13.5 embryo pool of ICR mice were plated at 1×10⁶ cells per 100 mmdish and incubated overnight. Next day, the cells were washed once withPBS and cultured in 10 ml of ES medium. Twenty-four h after incubation,the supernatant of MEF culture was collected, filtered through a 0.22 μmpore-size filter, and stored at −20° C. until use.

Plasmid Construction

The open reading frame of human OCT3/4 was amplified by RT-PCR andcloned into pCR2.1-TOPO. An EcoRI fragment of pCR2.1-hOCT3/4 wasintroduced into the EcoRI site of pMXs retroviral vector. Todiscriminate each experiment, a 20-bp random sequence, designated N₂₀barcode, was introduced into the NotI/SalI site of Oct3/4 expressionvector. A unique barcode sequence was used in each experiment to avoidinter-experimental contamination. The open reading frames of human SOX2,KLF4, and c-MYC were also amplified by RT-PCR and subcloned intopENTR-D-TOPO (Invitrogen). All of the genes subcloned into pENTR-D-TOPOwere transferred to pMXs by using the Gateway cloning system(Invitrogen), according to the manufacturer's instructions. Mouse Slc7a1ORF was also amplified, subcloned into pENTR-D-TOPO, and transferred topLenti6/UbC/V5-DEST (Invitrogen) by the Gateway system. The regulatoryregions of the human OCT3/4 gene and the REX1 gene were amplified by PCRand subcloned into pCRXL-TOPO (Invitrogen). For phOCT4-Luc andphREX1-Luc, the fragments were removed by KpnI/BglII digestion frompCRXL vector and subcloned into the KpnI/BglII site of pGV-BM2. ForpPolII-Luc, an AatII (blunted)/NheI fragment of pQBI-polII was insertedinto the KpnI (blunted)/NheI site of pGV-BM2. All of the fragments wereverified by sequencing. Primer sequences are shown in TABLE 13.

TABLE 13 Primer Sequences SEQ ID Primer NO: Sequence (5′ to 3′)Applications hOCT3/4-S944 26 CCC GAG GGC CCC ATT TTG GTA CC OCT3/4 TgPCR hSOX2-S691 27 GGC ACC CCT GGC ATG GCT CTT GGC TC SOX2 Tg PCRhKLF4-S1128 28 ACG ATC GTG GCC CCG GAA AAG GAC C KLF4 endo and Tg PCRhMYC-S1011 29 CAA CAA CCG AAA ATG CAC CAG CCC c-MYC Tg PCR CAGpMXs-AS3200 30 TTA TCG TCG ACC ACT GTG CTG CTG Tg PCR pMXs-L3205 31 CCCTTT TTC TGG AGA CTA AAT AAA Tg PCR hOCT3/4-S1165 32 GAC AGG GGG AGG GGAGGA GCT AGG Endo OCT3/4 hOCT3/4-AS1283 33 CTT CCC TCC AAC CAG TTG CCCCAA AC RT-PCR hSOX2-S1430 34 GGG AAA TGG GAG GGG TGC AAA AGA GG EndoSOX2 hSOX2-AS1555 35 TTG CGT GAG TGT GGA TGG GAT TGG TG RT-PCRECAT4-macaca-968S 36 CAG CCC CGA TTC TTC CAC CAG TCC C NANOG RT-PCRECAT4-macaca- 37 CGG AAG ATT CCC AGT CGG GTT CAC C 1334AS hGDF3-S243 38CTT ATG CTA CGT AAA GGA GCT GGG GDF3 RT-PCR hGDF3-AS850 39 GTG CCA ACCCAG GTC CCG GAA GTT hREXI-RT-U 40 CAG ATC CTA AAC AGC TCG CAG AAT REX1RT-PCR hREXI-RT-L 41 GCG TAC GCA AAT TAA AGT CCA GA hFGF4-RT-U 42 CTACAA CGC CTA CGA GTC CTA CA FGF4 RT-PCR hFGF4-RT-L 43 GTT GCA CCA GAA AAGTCA GAG TTG hpH34-S40 44 ATA TCC CGC CGT GGG TGA AAG TTC ESG1 RT-PCRhpH34-AS259 45 ACT CAG CCA TGG ACT GGA GCA TCC hECAT15-1-S532 46 GGA GCCGCC TGC CCT GGA AAA TTC DPPA4 RT-PCR hECAT15-1-AS916 47 TTT TTC CTG ATATTC TAT TCC CAT hECAT15-2-S85 48 CCG TCC CCG CAA TCT CCT TCC ATC DPPA2RT-PCR hECAT15-2-AS667 49 ATG ATG CCA ACA TGG CTC CCG GTG hTERT-S3234 50CCT GCT CAA GCT GAC TCG ACA CCG TG hTERT RT-PCR hTERT-AS3713 51 GGA AAAGCT GGC CCT GGG GTG GAG C hKLF4-AS1826 52 TGA TTG TAG TGC TTT CTG GCTGGG CTC C Endo KLF4 RT-PCR hMYC-S253 53 GCG TCC TGG GAA GGG AGA TCC GGAGC Endo c-MYC hMYC-AS555 54 TTG AGG GGC ATC GTC GCG GGA GGC TG RT-PCRhMSX1-S665 55 CGA GAG GAC CCC GTG GAT GCA GAG MSX1 RT-PCR hMSX1-AS938 56GGC GGC CAT CTT CAG CTT CTC CAG hBRACHYURY- 57 GCC CTC TCC CTC CCC TCCACG CAC AG BRACHYURY/T S1292 RT-PCR hBRACHYURY- 58 CGG CGC CGT TGC TCACAG ACC ACA GG AS1540 hGFAP-S1040 59 GGC CCG CCA CTT GCA GGA GTA CCA GGGFAP RT-PCR hGFAP-AS1342 60 CTT CTG CTC GGG CCC CTC ATG AGA CGhPAX6-S1206 61 ACC CAT TAT CCA GAT GTG TTT GCC CGA G PAX6 RT-PCRhPAX6-AS1497 62 ATG GTG AAG CTG GGC ATA GGC GGC AG hFOXA2-S208 63 TGGGAG CGG TGA AGA TGG AAG GGC AC FOXA2 RT-PCR hFOXA2-AS398 64 TCA TGC CAGCGC CCA CGT ACG AGG AC hSOX17-S423 65 CGC TTT CAT GGT GTG GGC TAA GGA CGSOX17 RT-PCR hSOX17-AS583 66 TAG TTG GGG TGG TCC TGC ATG TGC TGhAADC-S1378 67 CGC CAG GAT CCC CGC TTT GAA ATC TG AADC RT-PCRhAADC-AS1594 68 TCG GCC GCC AGC TCT TTG ATG TGT TC hChAT-S1360 69 GGAGGC GTG GAG CTC AGC GAC ACC ChAT RT-PCR hChAT-AS1592 70 CGG GGA GCT CGCTGA CGG AGT CTG hMAP2-S5401 71 CAG GTG GCG GAC GTG TGA AAA TTG MAP2RT-PCR AGA GTG hMAP2-AS5587 72 CAC GCT GGA TCT GCC TGG GGA CTG TGhDAT-S1935 73 ACA GAG GGG AGG TGC GCC AGT TCA CG SLC6A3/DAT hDAT-AS220774 ACG GGG TGG ACC TCG CTG CAC AGA TC RT-PCR hLMX1B-S770 75 GGC ACC AGCAGC AGC AGG AGC AGC AG LMX1B RT-PCR hLMXIB-AS1020 76 CCA CGT CTG AGG AGCCGA GGA AGC AG hMYL2A-S258 77 GGG CCC CAT CAA CTT CAC CGT CTT CC MYL2ART-PCR hMYL2A-AS468 78 TGT AGT CGA TGT TCC CCG CCA GGT CC hTnTc-S524 79ATG AGC GGG AGA AGG AGC GGC AGA AC TnTc RT-PCR hTnTc-AS730 80 TCA ATGGCC AGC ACC TTC CTC CTC TC hMEF2C-S1407 81 TTT AAC ACC GCC AGC GCT CTTCAC CTT G MEF2C RT-PCR hMEF2C-AS1618 82 TCG TGG CGC GTG TGT TGT GGG TATCTC G hMYHCB-S5582 83 CTG GAG GCC GAG CAG AAG CGC AAC G MYHCB RT-PCRhMYHCB-AS5815 84 GTC CGC CCG CTC CTC TGC CTC ATC C dT₂₀ 85 TTT TTT TTTTTT TTT TTT TT Reverse transcription hMYC-S857 86 GCC ACA GCA AAC CTCCTC ACA GCC CAC Southern blot probe hMYC-AS1246 87 CTC GTC GTT TCC GCAACA AGT CCT CTT C hOCT3/4-S 88 CAC CAT GGC GGG ACA CCT GGC TTC AG OCT3/4cloning hOCT3/4-AS 89 ACC TCA GTT TGA ATG CAT GGG AGA GC hSOX2-S 90 CACCAT GTA CAA CAT GAT GGA GAC SOX2 cloning GGA GCT G hSOX2-AS 91 TCA CATGTG TGA GAG GGG CAG TGT GC hKLF4-S 92 CAC CAT GGC TGT CAG TGA CGC GCTGCT KLF4 cloning CCC hKLF4-AS 93 TTA AAA ATG TCT CTT CAT GTG TAA GGC GAGhMYC-S 94 CAC CAT GCC CCT CAA CGT TAG CTT CAC c-MYC cloning CAA hMYC-AS95 TCA CGC ACA AGA GTT CCG TAG CTG TTC AAG Slc7a1-S 96 CAC CAT GGG CTGCAA AAA CCT GCT CGG Mouse Slc7a1 Slc7a1-AS 97 TCA TTT GCA CTG GTC CAAGTT GCT GTC cloning hREX1-pro5K-S 98 ATT GTC GAC GGG GAT TTG GCA GGG TCAPromoter cloning CAG GAC hREXx1-pro5K-AS 99 CCC AGA TCT CCA ATG CCA CCTCCT CCC AAA CG hOCT3/4-pro5K-S 100 CACTCG AGG TGG AGG AGC TGA GGG CACTGT GG hOCT3/4-pro5K-AS 101 CAC AGA TCT GAA ATG AGG GCT TGC GAA GGG ACmehREX1-F1-S 102 GGT TTA AAA GGG TAA ATG TGA TTA TAT Bisulfitesequencing TTA mehREX1-F1-AS 103 CAA ACT ACA ACC ACC CAT CAA C mehOCT3/4F2-S 104 GAG GTT GGA GTA GAA GGA TTG TTT TGG TTT mehOCT3/4 F2-AS 105 CCCCCC TAA CCC ATC ACC TCC ACC ACC TAA mehNANOG-FI-S 106 TGG TTA GGT TGGTTT TAA ATT TTT G mehNANOG-FI-AS 107 AAC CCA CCC TTA TAA ATT CTC AAT TA

Lentivirus Production and Infection

293FT cells (Invitrogen) were plated at 6×10⁶ cells per 100 mm dish, andincubated overnight. Cells were transfected with 3 μg ofpLenti6/UbC-Slc7a1 along with 9 μg of Virapower packaging mix byLipofectamine 2000 (Invitrogen), according to the manufacturer'sinstructions. Forty-eight hours after transfection, the supernatant oftransfectant was collected and filtered through a 0.45 μm pore-sizecellulose acetate filter (Whatman). Human fibroblasts were seeded at8×10⁵ cells per 100 mm dish 1 day before transduction. The medium wasreplaced with virus-containing supernatant supplemented with 4 μg/mlpolybrene (Nacalai Tesque), and incubated for 24 hours.

Retroviral Infection and iPS Cell Generation

PLAT-E packaging cells were plated at 8×10⁶ cells per 100 mm dish andincubated overnight. Next day, the cells were transfected with pMXsvectors with Fugene 6 transfection reagent (Roche). Twenty-four hoursafter transfection, the medium was collected as the firstvirus-containing supernatant and replaced with a new medium, which wascollected after 24 hours as the second virus-containing supernatant.Human fibroblasts expressing mouse Slc 7a1 gene were seeded at 8×10⁵cells per 100 mm dish 1 day before transduction. The virus-containingsupernatants were filtered through a 0.45-μm pore-size filter, andsupplemented with 4 μg/ml polybrene. Equal amounts of supernatantscontaining each of the four retroviruses were mixed, transferred to thefibroblast dish, and incubated overnight. Twenty-four hours aftertransduction, the virus-containing medium was replaced with the secondsupernatant. Six days after transduction, fibroblasts were harvested bytrypsinization and re-plated at 5×10⁴ cells per 100-mm dish on an SNLfeeder layer. Next day, the medium was replaced with hES mediumsupplemented with 4 ng/ml bFGF. The medium was changed every other day.Thirty days after transduction, colonies were picked up and transferredinto 0.2 ml of hES cell medium. The colonies were mechanicallydissociated to small clumps by pipeting up and down. The cell suspensionwas transferred on SNL feeder in 24-well plates. This stage was definedas passage 1.

RNA Isolation and Reverse Transcription

Total RNA was purified with Trizol reagent (Invitrogen) and treated withTurbo DNA-free kit (Ambion) to remove genomic DNA contamination. Onemicrogram of total RNA was used for reverse transcription reaction withReverTraAce-α (Toyobo, Japan) and dT₂₀ primer, according to themanufacturer's instructions. PCR was performed with ExTaq (Takara,Japan). Quantitative PCR was performed with Platinum SYBR Green qPCRSupermix UDG (Invitrogen) and analyzed with the 7300 real-time PCRsystem (Applied Biosystems). Primer sequences are shown in TABLE 13.

Alkaline Phosphatase Staining and Immunocytochemistry

Alkaline phosphatase staining was performed using the Leukocyte AlkalinePhosphatase kit (Sigma). For immunocytochemistry, cells were fixed withPBS containing 4% paraformaldehyde for 10 min at room temperature. Afterwashing with PBS, the cells were treated with PBS containing 5% normalgoat or donkey serum (Chemicon), 1% bovine serum albumin (BSA, Nacalaitesque), and 0.1% Triton X-100 for 45 min at room temperature. Primaryantibodies included SSEA1 (1:100, Developmental Studies Hybridoma Bank),SSEA3 (1:10, a kind gift from Dr. Peter W. Andrews), SSEA4 (1:100,Developmental Studies Hybridoma Bank), TRA-2-49/6E (1:20, DevelopmentalStudies Hybridoma Bank), TRA-1-60 (1:50, a kind gift from Dr. Peter W.Andrews), TRA-1-81 (1:50, a kind gift from Dr. Peter W. Andrews), Nanog(1:20, AF1997, R&D Systems), βIII-tubulin (1:100, CB412, Chemicon),glial fibrillary acidic protein (1:500, Z0334, DAKO), α-smooth muscleactin (pre-diluted, N1584, DAKO), desmin (1:100, RB-9014, Lab Vision),vimentin (1:100, SC-6260, Santa Cruz), α-fetoprotein (1:100, MAB1368,R&D Systems), tyrosine hydroxylase (1:100, AB 152, Chemicon). Secondaryantibodies used were cyanine 3 (Cy3)-conjugated goat anti-rat IgM(1:500, Jackson Immunoresearch), Alexa546-conjugated goat anti-mouse IgM(1:500, Invitrogen), Alexa488-conjugated goat anti-rabbit IgG (1:500,Invitrogen), Alexa488-conjugated donkey anti-goat IgG (1:500,Invitrogen), Cy3-conjugated goat anti-mouse IgG (1:500, Chemicon), andAlexa488-conjugated goat anti-mouse IgG (1:500, Invitrogen). Nuclei werestained with 1 μg/ml Hoechst 33342 (Invitrogen).

In Vitro Differentiation

For EB formation, human iPS cells were harvested by treating withcollagenase IV. The clumps of the cells were transferred topoly(2-hydroxyethyl methacrylate)-coated dish in DMEM/F12 containing 20%knockout serum replacement (KSR, Invitrogen), 2 mM L-glutamine, 1×10⁻⁴ Mnonessential amino acids, 1×10⁻⁴ M 2-mercaptoethanol (Invitrogen), and0.5% penicillin and streptomycin. The medium was changed every otherday. After 8 days as a floating culture, EBs were transferred togelatin-coated plate and cultured in the same medium for another 8 days.Co-culture with PA6 was used for differentiation into dopaminergicneurons. PA6 cells were plated on gelatin-coated 6-well plates andincubated for 4 days to reach confluence. Small clumps of iPS cells wereplated on PA6-feeder layer in Glasgow minimum essential medium(Invitrogen) containing 10% KSR (Invitrogen), 1×10⁻⁴ M nonessentialamino acids, 1×10⁻⁴ M 2-mercaptoethanol (Invitrogen), and 0.5%penicillin and streptomycin. For cardiomyocyte differentiation, iPScells were maintained on Matrigel-coated plate in MEF-CM supplementedwith 4 ng/ml bFGF for 6 days. The medium was then replaced with RPMI1640(Invitrogen) plus B27 supplement (Invitrogen) medium (RPMI/B27),supplemented with 100 ng/ml human recombinant activin A (R & D Systems)for 24 hours, followed by 10 ng/ml human recombinant bone morphologenicprotein 4 (BMP4, R&D Systems) for 4 days. After cytokine stimulation,the cells were maintained in RPMI/B27 without any cytokines. The mediumwas changed every other day.

Bisulfite Sequencing

Genomic DNA (1 μg) was treated with CpGenome DNA modification kit(Chemicon), according to the manufacturer's recommendations. Treated DNAwas purified with QIAquick column (QIAGEN). The promoter regions of thehuman Oct3/4, Nanog, and Rex1 genes were amplified by PCR. The PCRproducts were subcloned into pCR2.1-TOPO. Ten clones of each sample wereverified by sequencing with the M13 universal primer. Primer sequencesused for PCR amplification were provided in TABLE 13.

Luciferase Assay

Each reporter plasmid (1 μg) containing the firefly luciferase gene wasintroduced into human iPS cells or HDF with 50 ng of pRL-TK (Promega).Forty-eight hours after transfection, the cells were lysed with 1×passive lysis buffer (Promega) and incubated for 15 min at roomtemperature. Luciferase activities were measured with a Dual-Luciferasereporter assay system (Promega) and Centro LB 960 detection system(BERTHOLD), according to the manufacturer's protocol.

Teratoma Formation

The cells were harvested by collagenase IV treatment, collected intotubes and centrifuged, and the pellets were suspended in DMEM/F12. Onequarter of the cells from a confluent 100 mm dish was injectedsubcutaneously to dorsal flank of a SCID mouse (CREA, Japan). Nine weeksafter injection, tumors were dissected, weighted, and fixed with PBScontaining 4% paraformaldehyde. Paraffin-embedded tissue was sliced andstained with hematoxylin and eosin.

Western Blotting

The cells at semiconfluent state were lysed with RIPA buffer (50 mMTris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% sodiumdeoxycholate, and 0.1% SDS), supplemented with protease inhibitorcocktail (Roche). The cell lysate of MEL-1 hES cell line was purchasedfrom Abcam. Cell lysates (20 μg) were separated by electrophoresis on 8%or 12% SDS-polyacrylamide gel and transferred to a polyvinylidinedifluoride membrane (Millipore). The blot was blocked with TBST (20 mMTris-HCl, pH 7.6, 136 mM NaCl, and 0.1% Tween-20) containing 1% skimmilk and then incubated with primary antibody solution at 4° C.overnight. After washing with TBST, the membrane was incubated withhorseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour atroom temperature. Signals were detected with Immobilon Westernchemiluminescent HRP substrate (Millipore) and LAS3000 imaging system(FUJIFILM, Japan). Antibodies used for western blotting were anti-Oct3/4(1:600, SC-5279, Santa Cruz), anti-Sox2 (1:2000, AB5603, Chemicon),anti-Nanog (1:200, R&D Systems), anti-Klf4 (1:200, SC-20691, SantaCruz), anti-c-Myc (1:200, SC-764, Santa Cruz), anti-E-cadherin (1:1000,610182, BD Biosciences), anti-Dppa4 (1:500, ab31648, Abcam), anti-FoxD3(1:200, AB5687, Chemicon), anti-telomerase (1:1000, ab23699, Abcam),anti-Sall4 (1:400, ab29112, Abcam), anti-LIN-28 (1:500, AF3757, R&Dsystems), anti-β-actin (1:5000, A5441, Sigma), anti-mouse IgG-HRP(1:3000, #7076, Cell Signaling), anti-rabbit IgG-HRP (1:2000, #7074,Cell Signaling), and anti-goat IgG-HRP (1:3000, SC-2056, Santa Cruz).

Southern Blotting

Genomic DNA (5 μg) was digested with BglII, EcoRI, and NcoI overnight.Digested DNA fragments were separated on 0.8% agarose gel andtransferred to a nylon membrane (Amersham). The membrane was incubatedwith digoxigenin (DIG)-labeled DNA probe in DIG Easy Hyb buffer (Roche)at 42° C. overnight with constant agitation. After washing, alkalinephosphatase-conjugated anti-DIG antibody (1:10,000, Roche) was added toa membrane. Signals were raised by CDP-star (Roche) and detected byLAS3000 imaging system.

Short Tandem Repeat Analysis and Karyotyping

The genomic DNA was used for PCR with Powerplex 16 system (Promega) andanalyzed by ABI PRISM 3100 Genetic analyzer and Gene Mapper v3.5(Applied Biosystems). Chromosomal G-band analyses were performed at theNihon Gene Research Laboratories, Japan.

Detection of Telomerase Activity

Telomerase activity was detected with a TRAPEZE telomerase detection kit(Chemicon), according to the manufacturer's instructions. The sampleswere separated by TBE-based 10% acrylamide non-denaturing gelelectrophoresis. The gel was stained with SYBR Gold (1:10,000,Invitrogen).

Example 21 Generation of Induced Pluripotent Stem Cells without Myc

The direct reprogramming of somatic cells is considered to provide anopportunity to generate patient- or disease-specific pluripotent stemcells. Such pluripotent stem (iPS) cells are induced from mousefibroblasts by the retroviral transduction of four transcriptionfactors, Oct3/4, Sox2, Klf4, and c-Myc (Takahashi et al., Cell126:663-76, 2006). Mouse iPS cells are indistinguishable from mouse EScells in many aspects and give rise to germline-competent chimeras(Wemig et al., Nature 448:318-24, 2007; Okita et al., Nature 448:313-17,2007; Maherali et al., Cell Stem Cell 1:55-70, 2007). It was noted abovethat each iPS clone contained 3-6 retroviral integrations for eachfactor. Thus, each clone had more than 20 retroviral integration sitesin total, which may increase the risk of tumorigenesis. In the case ofmouse iPS cells, ˜20% of chimera mice and their offspring derived fromiPS cells developed tumors (Okita et al., Nature 448:313-17, 2007). Inparticular, it was found that the reactivation of the c-Myc retrovirusresults in an increased incidence of tumor formation in the chimeras andprogeny mice generated with mouse iPS cells, thus hindering the clinicalapplication of this technology (Okita et al., Nature 448:313-17, 2007).Therefore, a modified protocol for the induction of iPS cells, whichdoes not require the Myc retrovirus was developed. With this newprotocol, significantly fewer non-iPS background cells were obtained.Furthermore, the iPS cells generated without Myc were constantly of highquality. These findings are important for the future clinicalapplication of this iPS cell technology.

Substitution of the Four Factors with Other Family Members

This study was initiated to examine whether the family proteins of thefour factors could also induce iPS cells. In other words, furtherinvestigations were performed to assess which family members of a givengene family could substitute for others as nuclear reprogrammingfactors. Mouse embryonic fibroblasts (MEF) containing aGFP-IRES-Puro^(r) transgene driven by the Nanog gene regulatory elementswere used (Okita et al., Nature 448:313-17, 2007). Nanog is specificallyexpressed in mouse ES cells and preimplantation embryos (Chambers etal., Cell 113: 643-55, 2003; Mitsui et al., Cell 113: 631-42, 2003) andcan serve as a selection marker during iPS cell induction. Byintroducing the aforementioned four factors, iPS cells are induced asGFP-expressing colonies. Nanog-selected iPS cells are indistinguishablefrom ES cells and have been shown to give rise to germline-competentchimeras (Wemig et al., Nature 448: 318-24, 2007; Okita et al., Nature448:313-17, 2007; Maherali et al., Cell Stem Cell 1:55-70, 2007).

Oct3/4 belongs to the Oct family transcription factors, which containthe POU domain (Ryan et al., Genes Dev 11: 1207-25, 1997). The closesthomologs of Oct3/4 are Oct1 and Oct6. Oct3/4, Oct1, or Oct6 wereintroduced together with the remaining three factors, into theNanog-reporter MEF by retroviruses. With Oct3/4, many GFP-positivecolonies were observed (FIG. 43A). In contrast, no GFP-positive colonieswere obtained with Oct1 or Oct6, thus indicating the inability of thesetwo homologs to induce iPS cells.

Sox2 belongs to the Sox (SRY-related HMG-box) transcription factors,characterized by the presence of the high mobility group (HMG) domain(Schepers et al., Dev Cell 3: 167-70, 2002). Sox1, Sox3, Sox7, Sox15,Sox17, and Sox18 were tested and GFP-positive colonies were obtainedwith Sox1. In addition, fewer GFP-positive colonies were obtained withSox3, Sox15, and Sox18 (FIG. 43A). Sox18, however, failed to expand thecells.

Klf4 belongs to Krüppel-like factors (Klfs), zinc-finger proteins thatcontain amino acid sequences that resemble those of the Drosophilaembryonic pattern regulator Krüppel (Dang et al., Int J Biochem CellBiol 32: 1103-21, 2000). Klf1, Klf2, and Klf5 were tested andGFP-expressing colonies with Klf2 were thus obtained (FIG. 43A). Klf1and Klf5 were also capable of inducing iPS cells, but with a lowerefficiency.

c-Myc has two related proteins, N-Myc and L-Myc (Adhikary et al., NatRev Mol Cell Biol 6: 635-45, 2005). GFP-positive colonies emerged withboth N-Myc and L-Myc (FIG. 43A). Therefore, some, but not all familyproteins of the four factors can induce iPS cells.

The family proteins were also tested for their ability to induce iPScells from MEFs in which βgeo was knocked into the Fbx15 locus (Tokuzawaet al., Mol Cell Biol 23: 2699-708, 2003). Similar results to those withthe Nanog-based selection were obtained: Sox2 could be replaced by Sox1and Sox3, Klf4 by Klf2, and c-Myc by N-Myc and L-Myc. The cellsgenerated by the family proteins were expandable and showed a morphologyindistinguishable from that of ES cells (not shown). They gave rise toteratomas in nude mice (FIG. 44). Therefore, some family proteins arecapable of inducing iPS cells from both Nanog-reporter MEF andFbx15-reporter MEF.

As has been stated above, iPS cell generation from somatic cells wasevaluated by optimizing retroviral transduction and subsequent cultureconditions. Furthermore, optimization would be useful for theapplication of this iPS cell technology to human cells, especially inclinical situations. Unexpectedly, a few ES cell-like and GFP-positivecolonies from Nanog-reporter MEF were obtained without any Mycretroviruses (FIG. 43A). This was in contrast to a previous study inwhich no GFP-positive colonies could be obtained without c-Myc (Okita etal., Nature 448:313-17, 2007). Consistent with the efforts towardoptimization, one difference between the two studies is the timing ofthe drug selection: In the previous study, puromycin selection wasinitiated seven days after the transduction, whereas in this experimentthe selection was started at 14 days. This suggests that iPS cellinduction without Myc is a slower process than that with Myc.Furthermore, as is further discussed herein, the omission of Mycresulted in a less efficient but more specific induction of iPS cells.

Myc Omission Results in More Specific iPS Cell Induction

To test whether iPS cell induction without Myc is a slower process thanthat with Myc, Nanog-reporter MEFs were transduced with either the fourfactors or three factors devoid of Myc, and then puromycin selection wasstarted seven, 14, or 21 days after the transduction (FIG. 43B). Withthe four factors, GFP-positive colonies were observed in all of theconditions. The colony numbers significantly increased when puromycinselection was delayed. Without Myc, no GFP-colonies were observed whenselection was initiated seven days after the transduction. In contrast,GFP-positive colonies did emerge even without Myc when selection wasstarted 14 or 21 days after the transduction. The colony numbers werefewer with the three factors than with the four factors in eachcondition. Nanog-selected iPS cells generated without Myc retrovirusesexpressed ES cell marker genes at comparable levels to those in ES cells(FIG. 45), and thus gave rise to adult chimeras when transplanted intoblastocysts (TABLE 14).

TABLE 14 Summary of blastocysts injections origin- injected iPS clonesgenotype* selection blastocysts born mice chimeras 142B-6 MEF-FB/gfpG418 39 7 3 142B-12 46 12 5 178B-1 MEF-Ng Puro 156 50 5 178B-2 142 43 17178B-5 60 20 5 178B-6 28 10 4 256H-4 TTF-ACTB- No 72 6 5 256H-13 DsRed96 8 5 256H-18 90 17 11 All iPS clones were induced with three factorsdevoid of Myc from MEF or TTF. *FB, Fbx15-βgeo reporter; Ng,Nanog-GFP-IRES-Puro^(r) reporter; gfp, CAG-EGFP

Another difference is that fewer GFP-negative colonies as well asbackground cells were observed with the three factors devoid of Myc thanwith the four factors (FIG. 43C). Therefore, the omission of Mycresulted in a less efficient but more specific induction of iPS cells.

It was also possible to generate a few iPS cells without Myc from MEFs,in which βgeo was knocked into the Fbx15 locus (Tokuzawa et al., Mol.Cell. Biol. 23:2699-708, 2003). (FIG. 46A). This is again in contrast tothe original report, in which no iPS cells were obtained without c-Myc(Takahashi et al., Cell 126:663-76, 2006). In the two experiments, G418selection was initiated with the same timing: three days after thetransduction. However, the colonies were selected 14-21 days after thetransduction in the previous report, whereas ˜30 days were required inthe current study. Another difference was that the retroviraltransfection efficiency was raised by preparing each of the four orthree factors separately in an independent Plat-E (Morita et al., GeneTher. 7:1063-66, 2000) plate in this study. In comparison to theoriginal work in which all the four factors were prepared in a singlePlat-E plate, a significant increase in the number of iPS cell colonieswas observed (not shown). This is consistent with the notion that iPScell induction without Myc is a slower and less efficient process thanthat with Myc.

Fbx15-selected iPS cells, which were generated with the four factors,express lower levels of ES-cell marker genes than ES cells (Takahashi etal., Cell 126:663-76, 2006). They cannot produce adult chimeras whenmicroinjected into blastocysts. In contrast, iPS cells generated withoutMyc expressed ES-cell marker genes at comparable levels to those in EScells even with the Fbx15 selection (FIG. 46B). Furthermore, adultchimeras were obtained with high iPS cell contribution from these cells(FIG. 46C, TABLE 14). No increased incidence of tumor formation wasobserved in these chimeras.

iPS Cell Induction in the Absence of Drug Selection

Next, it was determined whether the omission of Myc would result inefficient isolation of iPS cells without drug selection. The four orthree factors were introduced into adult tail tip fibroblasts (TTF)containing the Nanog reporter, but puromycin selection was not applied.DsRed retrovirus was transduced together with the four or three factorsto visualize transduced cells. Thirty days after the retroviraltransduction, the dishes transduced with the four factors were coveredwith numerous GFP-negative colonies and background cells (FIG. 47A,TABLE 15).

TABLE 15 Summary of experiments (Nanog-GFP reporter TTF, withoutselection) Experiment Cell Total picked Number Factors seeded coloniesup established 220 4   5 × 10⁴ many (107) 26 (24) 25 (22) 256 4   5 ×10⁴ many (4) 3 3.5 × 10⁵  7 (4) 7 (4) 6 (5) 272 4 5.4 × 10⁴ many (132) 6(6) 5 (4) 3 3.1 × 10⁵ 21 (8) 4 (4) 2 (2) 309 4 2.3 × 10⁴ many (424) 39.6 × 10⁵ 43 (24) Numbers in parentheses indicate number of colonies orclones that were positive for GFP. The ratios of the retroviruses,Oct3/4, Sox2, Klf4, (c-Myc), and DsRed, were 1:1:1:(1):4 in experiment256 and 1:1:1:(1):1 in experiments 272 and 309. In experiment 220, DsRedwas not introduced.

Using fluorescent microscopy, small portions of these colonies (4, 132,and 424 colonies in three independent experiments) were foundGFP-positive. Of note, the GFP-positive colonies were negative forDsRed, which was consistent with the retroviral silencing observed inNanog-selected iPS cells (Okita et al., Nature 448:313-17, 2007). Incontrast, with the three factors devoid of Myc, a small number (7, 21,and 43 in three independent experiments) of discrete colonies wereobserved with few background cells. Approximately a half of themexpressed GFP in a patchy manner. DsRed was only detected in a smallportion of some colonies, indicating that it was largely silenced. Nooverlap was observed between GFP and DsRed. Most of these colonies wereexpandable and produced iPS cells, which became positive for GFP andnegative for DsRed at passage 2. Therefore, the omission of c-Mycresulted in more specific generation of iPS cells, in which Nanog-GFP isactivated whereas the retroviruses are silenced.

Next, generation of iPS cells was attempted from adult TTF that did nothave selection markers, but had the DsRed transgene driven by aconstitutively active promoter (Vintersten et al. Genesis 40:241-46,2004). The four factors or the three factors devoid of Myc wereintroduced. In addition, a GFP retrovirus was introduced to monitorsilencing. After 30 days without drug selection, ˜1000 colonies emergedfrom 0.5×10⁵ cells transduced with the four factors. Most of them werepositive for GFP, indicating that retroviral silencing did not takeplace in these cells. In contrast, only 16 colonies (FIG. 47B) emergedfrom 3.5×10⁵ cells transduced with the three factors devoid of Myc. Mostof these colonies express no GFP, while the remaining expressed GFP insmall portions. All of these colonies were expandable and showed iPS- orES-like morphology at the second passage. They were all negative forGFP, thus indicating retroviral silencing. RT-PCR showed that thesecells expressed ES cell marker genes at comparable levels to those in EScells (FIG. 47C). In addition, RT-PCR confirmed the retroviral silencingof Klf4 and the absence of the Myc transgene in iPS cells generated withthe three factors. Furthermore, when transplanted into blastocysts,these cells gave rise to chimeras (FIG. 47D, TABLE 14). Therefore, byomitting Myc, good iPS cells can be efficiently generated from adult TTFwithout drug selection. These findings should be useful for theapplication of this iPS cell technology to human cells, especially inclinical situations.

Induction of Human iPS Cells without Myc Retroviruses

FIGS. 48(A)-(C) show induction of human iPS cells without Mycretroviruses. The retroviruses for Oct3/4, Sox2 and Klf4 were introducedinto BJ fibroblasts (246G) or HDF (253G). After 30 days, a few hEScell-like colonies emerged. These cells were expandable and showed hEScell-like morphology (FIG. 48(A)). Results were obtained for theexpression of ES cell marker genes in human iPS cells derived from HDFwithout Myc retroviruses (253G) or with Myc (253F) (FIG. 48(B)), as wereresults for embryoid body-mediated differentiation of human iPS cellsgenerated without Myc retroviruses (FIG. 48(C)).

Experimental Procedures for Example 21

Plasmid construction. The coding regions of family genes were amplifiedby RT-PCR with primers listed in TABLE 16, subcloned into pDONR201 orpENTR-D-TOPO (Invitrogen), and recombined with pMXs-gw by the LRreaction (Invitrogen).

TABLE 16 Primers used for cloning of the family factors Genes SequencesSEQ ID NO: Sox1 CAC CAT GTA CAG CAT GAT GAT GGA GAC CGA CCT 108 CTA GATATG CGT CAG GGG CAC CGT GC 109 Sox3 CAC CAT GTA CAG CCT GCT GGA GAC TGAACT CAA G 110 TCA GAT GTG GGT CAG CGG CAC CGT TCC ATT 111 Sox7 CAC CTCGGC CAT GGC CTC GCT GCT GGG 112 CTC CAT TCC TCC AGC TCT ATG ACA CAC 113Sox15 CAC CAT GGC GCT GAC CAG CTC CTC ACA A 114 TTA AAG GTG GGT TAC TGGCAT GGG 115 Sox17 CAC CAG AGC CAT GAG CAG CCC GGA TG 116 CGT CAA ATG TCGGGG TAG TTG CAA TA 117 Sox18 CAC CAT GCA GAG ATC GCC GCC CGG CTA CG 118CTA GCC TGA GAT GCA AGC ACT GTA ATA GAC 119 Oct1 CAC CAT GAA TAA TCC ATCAGA AAC CAA T 120 GCT CTG CAC TCA GCT CAC TGT GCC 121 Oct6 CAC CAT GGCCAC CAC CGC GCA GTA TCT G 122 GGA ACC CAG TCC GCA GGG TCA CTG 123 Klf1CAC CAT GAG GCA GAA GAG AGA GAG GAG GC 124 TCA GAG GTG ACG CTT CAT GTGCAG AGC TAA 125 Klf2 CAC CAT GGC GCT CAG CGA GCC TAT CTT GCC 126 CTA CATATG TCG CTT CAT GTG CAA GGC CAG 127 Klf5 CAC CAT GCC CAC GCG GGT GCT GACCAT G 128 TCG CTC AGT TCT GGT GGC GCT TCA 129 L-MycWT CAC CAT GGA CTTCGA CTC GTA TCA GCA CTA TTT C 130 TTA GTA GCC ACT GAG GTA CGC GAT TCTCTT 131 N- CAC CAT GCC CAG CTG CAC CGC GTC CAC CAT 132 MycWT TTA GCA AGTCCG AGC GTG TTC GAT CT 133

Retroviral transduction. pMXs-based retroviral vectors were transfectedinto Plat-E cells (Morita et al., Gene Ther. 7:1063-66, 2000) usingFugene 6 reagents (Roche) according to manufacturer's instruction.Twenty-four hours after transfection, the medium was replaced. After 24hours, virus-containing supernatant were used for retroviral infection.In a “mixed” protocol, the mixture of plasmids for the four factors wastransfected into a single dish of Plat-E cells. In a “separate” method,each plasmid was transfected into separate dishes of Plat-E cells.Virus-containing supernatant was mixed prior to transduction.Significantly higher transduction efficiency was observed with theseparate method.

Induction of iPS cells with drug selection. The induction of iPS cellswas performed as previously described (Takahashi et al., Cell126:663-76, 2006; Okita et al., Nature 448:313-17, 2007) with somemodifications. Briefly, MEFs, which contained either theNanog-GFP-IRES-Puro^(r) reporter or the Fbx15-βgeo reporter, or both,were seeded at 1.3 and 8.0×10⁵ cells/well in 6-well plates and 100 mmdish, respectively, with SNL feeder cells (McMahon et al., Cell62:1073-85, 1990). The transduced cells were cultivated with ES mediumcontaining LIF (Meiner et al., Proc. Natl. Acad. Sci. U.S.A.93:14041-46. (1996). Selection with G418 (300 μg/ml) or puromycin (1.5μg/ml) was started as indicated. Twenty-five to 30 days aftertransduction, the number of colonies was recorded. Some colonies werethen selected for expansion.

iPS cells induction without drug selection. TTFs were isolated fromadult Nanog-reporter mice or adult DsRed-transgenic mice (Vintersten etal., Genesis 40:241-46, 2004). Retroviral-containing supernatant wasprepared in the separated method. For the four-factor transduction,retrovirus-containing supernatants for Klf4, c-Myc, Oct3/4, Sox2 andDsRed, were mixed with the ratio of 1:1:1:1:4. When the three factorswere transduced, retrovirus-containing supernatants for Klf4, Oct3/4,Sox2, Mock, and DsRed were mixed with the ratio of 1:1:1:1:4. With DsRedtransgenic mice, the GFP retrovirus was used instead of DsRed. Fortransfection, TTFs were seeded at 8.0×10⁵ cells per 100-mm dishes, whichdid not have feeder cells. TTFs were incubated in thevirus/polybrene-containing supernatants for 24 hours. Four days aftertransduction, TTFs transduced with the three factors were reseeded at3.5×10⁵ cells per 100-mm dishes with SNL feeder cells and cultured withES medium. TTFs transduced with the four factors were re-seeded at0.5×10⁵ cells per 100-mm dishes with feeder cells. Thirty to 40 daysafter transduction, the colonies were selected for expansion.

Characterization of iPS cells. RT-PCR and teratoma formation wereperformed as previously described. For the chimera experiments, 15-20iPS cells were injected into BDFI-derived blastocysts, which were thentransplanted into the uteri of pseudo-pregnant mice.

Example 22 Establishment of Human iPS Cells from Epithelial Cells withSix Factors

Among the nuclear reprogramming factors disclosed herein is a nuclearreprogramming factor comprising one or more gene products from thefollowing genes: Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 (NCBIaccession number NM_(—)145833 (mouse) and NM_(—)024674 (human)).Establishment of induced pluripotent stem cells was performed withcombinations of these gene products. The results are shown in TABLE 17.

TABLE 17 Summary of Experiments with Six Factors Day 23 non Day 29 Day23 ES like ES like non ES like ES like non ES like ES like 6F 59 39 16742 16 27 -L 49 5 53 14 -N 220 11 216 47 -M 2 0 15 0 -O 0 0 0 0 -S 491 0489 0 -K 61 0 51 0 -KS 1206 0 1305 0 -KO 0 0 0 0 -KM 0 0 0 0 0 0 -KN 510 57 0 -KL 28 0 41 0 -SO 0 0 0 0 -SM 0 0 0 0 -SN 188 0 171 0 -SL 112 0136 0 -OM 0 0 0 0 -ON 0 0 0 0 -OL 0 0 0 0 -MN 3 0 8 0 -ML 0 0 0 0 -NL 981 119 9 17 6 GFP 0 0 0 0 KO 0 0 0 0 KS 0 0 0 0

6×10⁶ 293FT cells were plated on 10 cm dish and cultured overnight, andthen transfected with 3 μg of pLenti6/UbC-Slc7a1 lentiviral vectortogether with 9 μg of Virapower packaging mix by Lipofectamine 2000(Invitrogen). After 24 hours, the culture medium was replaced with afresh medium. After 20 hours, the culture supernatant was collected andfiltrated through 0.45-μm pore-size cellulose acetate filter (Whatman).5×10⁵ epithelial cells were prepared on the previous day. To the dishwhich the culture supernatant was removed from, the aforementionedfiltrated culture supernatant containing viruses and 4 μg/ml polybrene(Nacalai Tesque) were added. Then, the cells were cultured for 24 hours.

In addition, 1.0×10⁶ Plat-E cells were plated on 6 cm dish and cultured.On the next day, the cells were transfected with 9.0 μg of pMX-basedretrovirus vector incorporating klf4, c-myc, oct3/4, sox2, nanog and/orLin-28 by using 27 μl of Fugene6 transfection reagent. After 24 hours,the culture medium was replaced with a fresh medium. On the next day,the culture supernatant of Plat-E cells was collected and filtratedthrough 0.45-μm pore-size cellulose acetate filter (Whatman). Seven daysafter lentivirus infection, epithelial cells were plated at 3.0×10⁵cells per 6 cm dish again, and the aforementioned culture supernatantcontaining retrovirus and polybrene were added thereto.

The term “6F” in TABLE 17 refers to the six factors (klf4, c-myc,oct3/4, sox2, nanog and Lin-28), the term “L” refers to Lin-28, the term“N” refers to nanog, the term “M” refers to c-Myc, the term “O” refersto Oct3/4, the term “S” refers to Sox2, and the term “K” refers to Klf4,respectively. The symbol “−” refers to colonies obtained by subtractingfrom the six factors those factors shown by the term subsequent to thesymbol “−”. For example, the term “−L” shows the colonies obtained withthe remaining five factors other than lin-28, and the term “−KS” showsthe colonies obtained with the remaining four factors other than Klf4and Sox2, respectively.

The numbers in TABLE 17 refer to the number of colonies. The term“non-ES like” refers to shows colonies having a non-ES cell likemorphology, and the term “ES like” refers to colonies having an ES likecell morphology.

Two experimental results are shown. The first experiment shows thenumber of colonies from cells introduced with various combinations offactors 23 days or 29 days after gene introduction and the secondexperiment shows the number of “6F,” “−KM,” and “−NL.” According tothese experimental results, the number of colonies from cellsnot-introduced with lin-28 such as “−L” was larger than that of celltransduced with lin-28, suggesting that Lin-28 plays an important roleto improve the efficiency of establishment of iPS cells.

In addition, iPS cell induction experiments were performed with sixfactors (Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28) and two differentcombinations of four factors (Klf4, c-Myc, Oct3/4 and Sox2, referred toas Y4F in FIG. 49; and Oct3/4, Sox2, Nanog, and Lin-28, referred to asT4F in FIG. 49). The second combination of four factors, T4F, is thesame combination as disclosed in Yu et al., Science 318:1917-1920, 2007.In these experiments, use of the six factors and the Y4F combination offour factors generated colonies having a similar morphology as ES-likecell colony, whereas the T4F combination generated no colonies having asimilar morphology as ES-like cell colony (FIG. 49).

Example 23 More Efficient iPS Cell Generation with Sall4

Experiments performed with mouse embryonic fibroblasts and adult humandermal fibroblasts showed that iPS cell induction with three factors(Klf4, Oct3/4, and Sox2) is more efficient when Sall4 is added to thecombination, that is when Klf4, Oct3/4, Sox2, and Sall4 are used (FIGS.50(A)-(C) and FIG. 51). More ES like colonies were also observed whenSall4 was added to the four factors (Klf4, Oct3/4, Sox2, and c-Myc) orthe three factors (Klf4, Oct3/4, Sox2) under the experimental conditionsused. These experiments show that addition of Sall4 to the nuclearreprogramming factor can improve iPS induction efficiency.

Kits of the Present Invention.

One aspect of the present invention includes kits designed for use inthe preparation and induction of iPS cells. Another aspect of theinvention comprises kits for the prevention or treatment of a medicalcondition or disease through the use of an NRF, an iPS cell or a cellderived from an iPS cell by induction of differentiation. It is also anobject of the present invention to provide compositions and methodsuseful for in vitro transfection, in vivo transfection, ex vivotransfection, in situ labeling, diagnostic tests, genetic therapy, genetherapy, treatment of medical conditions, and the creation of transgenicanimals. One aspect of the present invention comprises kits designed forin vitro transfection, in vivo transfection, ex vivo transfection, insitu labeling, diagnostic tests, genetic therapy, gene therapy,treatment of medical conditions, and the creation of transgenic animals.Accordingly, the present invention includes a composition comprising oneor more NRFs, one or more iPS cells, one or more cells derived from aniPS cell or cells, and combinations thereof.

UTILITY AND PRACTICAL APPLICATIONS

By using the nuclear reprogramming factor provided by the presentinvention, reprogramming of differentiated cell nuclei can beconveniently and highly reproducibly induced without using embryos or EScells, and induced pluripotent stem cells as undifferentiated cellshaving differentiation ability, pluripotency and growth ability similarto those of ES cells can be established.

Uses of the induced pluripotent stem cells prepared by the method of thepresent invention are not particularly limited. The cells can be usedfor any experiments and research conducted with ES cells, therapeutictreatments utilizing ES cells and the like. For example, desireddifferentiated cells (e.g., nerve cells, cardiac muscle cells, hemocytecells and the like) can be derived by treating induced pluripotent stemcells obtained by the method of the present invention with retinoicacid, growth factors such as EGF, glucocorticoid or the like, and stemcell therapy based on cellular auto-transplantation can be achieved byreturning the differentiated cells obtained as described above to thepatient. However, uses of the induced pluripotent stem cells of thepresent invention are not limited to the aforementioned specificembodiments.

Thus, the present invention has enabled the generation of iPS cells fromadult human dermal fibroblasts and other human somatic cells, which areindistinguishable from human ES cells in their differentiation potentialin vitro and in teratomas. Furthermore, the instant invention allows forthe generation of patient- and disease-specific pluripotent stem cells.Even with the presence of retroviral integration, human iPS cells areuseful for understanding disease mechanisms, drug screening, andtoxicology. For example, hepatocytes derived from iPS cells with variousgenetic and disease backgrounds can be utilized in predicting livertoxicity of drug candidates. Human iPS cells may overcome the ethicalissues that hES cells confront.

Reference is made to the following documents: Takahashi, et al. Cell131:861-872, 2007; Nakagawa et al. Nature Biotechnology 26(1):101-106,2008; Takahashi et al., Cell 126: 663-676, 2006; Okita et al., Nature448:313-17, 2007; Takahashi, et al. Nature Protocols 2(12):3081-89,2007; and the supplementary figures and data associated with thesedocuments, all of which are incorporated by reference herein in theirentireties.

Although the present invention has been described in considerable detailwith regard to certain versions thereof, other versions are possible,and alterations, permutations, and equivalents of the versions shownwill become apparent to those skilled in the art upon a reading of thespecification and study of the drawings. Also, the various features ofthe versions herein can be combined in various ways to provideadditional versions of the present invention. Furthermore, certainterminology has been used for the purposes of descriptive clarity, andnot to limit the present invention. Therefore, any appended claimsshould not be limited to the description of the preferred versionscontained herein and should include all such alterations, permutations,and equivalents as fall within the true spirit and scope of the presentinvention.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the methods of the presentinvention can be carried out with a wide and equivalent range ofconditions, formulations, and other parameters without departing fromthe scope of the invention or any embodiments thereof.

The attached Sequence Listing includes those sequences disclosed inPCT/JP2006/324881, which is incorporated by reference herein in itsentirety.

1. A method for preparing an induced pluripotent stem cell by nuclearreprogramming of a somatic cell from a mammalian species, comprising: a)introducing into the somatic cell one or more retroviral vectorscomprising a gene encoding Oct3/4, a gene encoding Klf4, a gene encodingc-Myc and a gene encoding Sox2 operably linked to a promoter; and b)culturing the transduced somatic cell on a fibroblast feeder layer orextracellular matrix in a cell media that supports growth of ES cells ofthe mammalian species, wherein one or more pluripotent cells areobtained.